Vinca Alkaloids Vinblastine Research Articles

The interaction of spongistatin 1 with tubulin

A tritiated derivative of the sponge-derived natural product spongistatin 1 was prepared, and its interactions with tubulin were examined. [3H]Spongistatin 1 was found to bind rapidly to tubulin at a single site (the low specific activity of the [3H]spongistatin 1, 0.75 Ci/mmol, prevented our defining an association rate), and the inability of spongistatin 1 to cause an aberrant assembly reaction was confirmed. Spongistatin 1 bound to tubulin very tightly, and we could detect no significant dissociation reaction from tubulin. The tubulin-[3H]spongistatin 1 complex did dissociate in 8 M urea, so there was no evidence for covalent bond formation. Apparent KD values were obtained by Scatchard analysis of binding data and by Hummel-Dreyer chromatography (3.5 and 1.1 μM, respectively). The effects of a large cohort of vinca domain drugs on the binding of [3H]spongistatin 1 to tubulin were evaluated. Compounds that did not cause aberrant assembly reactions (halichondrin B, eribulin, maytansine, and rhizoxin) caused little inhibition of [3H]spongistatin 1 binding. Little inhibition also occurred with the peptides dolastatin 15, its active pentapeptide derivative, vitilevuamide, or diazonamide A, nor with the vinca alkaloid vinblastine. Strong inhibition was observed with dolastatin 10, hemiasterlin, and cryptophycin 1, all of which cause aberrant assembly reactions that might actually mask the spongistatin 1 binding site. Spongistatin 5 was found to be a competitive inhibitor of [3H]spongistatin 1 binding, with an apparent Ki of 2.2 μM. We propose that the strong picomolar cytotoxicity of spongistatin 1 probably derives from its extremely tight binding to tubulin.

Abstract 1403: Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss

Abstract Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC is an aggressive subtype of breast cancer that lacks the expression of estrogen and progesterone receptors and HER2 protein. This lack of functioning receptors results in limited treatment options, usually resulting in the use of taxanes, platinums, and anthracyclines. Although TNBC often starts as being sensitive to traditional chemotherapies, tumor relapse and/or resistance often develops. Paclitaxel (PTX) is a commonly used taxane for TNBC, well known to induce mitotic arrest through stabilization of microtubules (MT) leading to an interference with the treadmilling function of MT through the activation of the spindle assembly checkpoint. We have previously shown that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor significantly increases resistance to PTX in APC knockdown (APCKD) cells generated in two human TNBC cell lines, MDA-MB-157 and MDA-MB-231. Given this striking PTX resistance, we were interested in seeing if resistance in APCKD can be seen using other non-taxane MT targeting agents. To accomplish this, we have used an epothilone (Epothilone B) and a vinca alkaloid (Vinblastine), that function by stabilizing and destabilizing the MTs, respectively. Our previous investigations demonstrated that APC loss results in increased CDK1 and CDK6, so these have been used as markers of cell cycle progression after drug treatment. In addition, changes in apoptosis were measured through the use of Annexin V and PI staining, and cleaved caspase 3 expression. Examined together, a better understanding of how APC loss influences chemoresistance in TNBC can lead to more efficient treatments for TNBC patients resistant to taxanes. Given the critical interaction between APC and MTs, this may be an avenue for targeted therapies to overcome taxane resistance. Citation Format: Pamela D. Arenas, Sara M. Maloney, Jenifer R. Prosperi. Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1403.

Abstract 3948: The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models

Abstract Checkpoint inhibitors (CIs) have shown an unprecedented clinical activity in a large variety of cancer types, but only in a fraction of patients. To increase the number of responders, preclinical models are studied to define effective combinatorial regimens and the best window of therapeutic opportunities. We recently described a synergy between anti-PD-1 and -PD-L1 CIs and several types and dosages of chemotherapy in immunocompetent models of triple negative breast cancer (TNBC), likely due to unique effects of chemotherapeutics over circulating and tumor-infiltrating, suppressive, antigen-presenting and effector immune cell populations (Orecchioni et al, Br J Cancer 2018). To find a high-order drug combination, we have further studied by multiparametric flow cytometry and single-cell transcriptomic a panel of different chemotherapy drugs (including microtubular poisons, alkylating agents, topoisomerase inhibitors and antimetabolites) used in vivo at different dosages and schedule. Our aim was to define a combination, dose and schedule able to promote a) dendritic cell maturation, b) release and c) uptake of cancer-associated antigens from targeted neoplastic cells, d) cross-priming between antigen-presenting cells (APCs) and T cells, e) NK cell activation, and f) inhibition of suppressor immune cell populations. Vinca alkaloids vinblastine and vinorelbine (V), at low-medium dosages, were the most effective in a), b), c) and d). After V administration, Tregs were reduced, and circulating and tumor-infiltrating APCs showed a “maturation to activation” program with increased expression of several antigens including CD40, CD80, and CD86. The alkylating agent cyclophosphamide (CTX) targeted Tregs, mobilized APC progenitors, activated and increased the number of circulating and intratumoral NK cells. These effects were significantly increased when CTX was administered in an intermittent fashion (every 6 days) at low-medium doses. As the APC-NK cell axis has been recently found to be pivotal in CI-mediated, anti-tumor cell immunity, our data suggested a preclinical trial in immunocompetent orthotopic models of metastatic (post-mastectomy) BALB mice injected with 4T1 or EMT-6 TNBC cells. Among a large panel of different combinations and dosages, the sequential administration of V, intermittent CTX and anti-PD-L1 was the only able to completely abrogate TNBC local and metastatic growth, and this effect was not observed in T and NK cell-depleted mice. Taken together with recent data from randomized, combinatorial studies in TNBC patients (Schmid et al, NEJM 2018), our findings suggest that CIs might be included in future combinatorial regimens aimed at improving the percentage of patients receiving a clinical benefit and prolonging the duration of this benefit. Citation Format: Stefania Orecchioni, Loredana Vecchi, Paolo Falvo, Lucilla Luzi, Patrizia Mancuso, Chiara Camisaschi, Francesco Bertolini. The sequential administration of vinca alkaloids and intermittent cyclophosphamide primes antigen-presenting and NK cells and significantly improves in vivo efficacy of anti-PD-L1 in triple-negative breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3948.

Metabolites of Vinca Alkaloid Vinblastine: Tubulin Binding and Activation of Nausea-Associated Receptors.

Vinblastine (VLB) is an antimitotic drug that binds to the vinca site of tubulin. The molecule possesses a high molecular weight and a complex chemical structure with many possibilities of metabolization. Despite advances in drug discovery research in reducing drug toxicity, the cause and mechanism of VLB-induced adverse drug reactions (ADRs) remains poorly understood. VLB is metabolized to at least 35 known metabolites, which have been identified and collected in this present work. This study also explores how VLB metabolites affect nausea-associated receptors such as muscarinic, dopaminergic, and histaminic. The metabolites have stronger binding interactions than acetylcholine (ACh) for muscarinic M1, M4, and M5 receptors and demonstrate similar binding profiles to that of the natural substrate, ACh. The affinities of VLB metabolites to dopaminergic and histaminic receptors, their absorption, distribution, metabolism, excretion, toxicity properties, and the superiority of VLB to ACh for binding to M5R, indicate their potential to trigger activation of nausea-associated receptors during chemotherapy with VLB. It has been shown that metabolite 20-hydroxy-VLB (metabolite 10) demonstrates a stronger binding affinity to the vinca site of tubulin than VLB; however, they have similar modes of action. VLB and metabolite 10 have similar gastric solubility (FaSSGF), intestinal solubility (FeSSIF), and log P values. Metabolite 10 has a more acceptable pharmacokinetic profile than VLB, a better gastric and intestinal solubility. Furthermore, metabolite 10 was found to be less bound to plasma proteins than VLB. These are desired and essential features for effective drug bioavailability. Metabolite 10 is not a substrate of CYP2D6 and thus is less likely to cause drug–drug interactions and ADRs compared to its parent drug. The hydroxyl group added upon metabolism of VLB suggests that it can also be a reasonable starting compound for designing the next generation of antimitotic drugs to overcome P-glycoprotein-mediated multidrug resistance, which is often observed with vinca alkaloids.

The Biological Activity of the Novel Vinca Alkaloids 4-chlorochablastine and 4-chlorochacristine.

Vinca alkaloids are important cancer drugs belonging to the class of antimitotic agents. The most commonly used substances are vinblastine and vincristine, other compounds are vinorelbine and vinflunine. All of them are very effective drugs but their use is limited by severe side-effects including neurotoxicity and bone marrow depression. Therefore, it is very important to develop novel vinca alkaloids with similar efficacy but lower toxicity. Here, we analyzed two new compounds, 4-chlorochablastine and 4-chlorochacristine, with regard to their biological activity. These novel compounds were applied to a leukemia cell line at clinically relevant concentrations. For comparison, the established vinca alkaloids vinblastine, vincristine, vinorelbine, and vinflunine were also tested. Both novel substances decreased cellular proliferation. Apoptosis was found to be increased using two different methods reflecting early and late apoptosis. Cell cycle analysis revealed a clear decrease in G1-cells and an increase in G2/M-cells indicating an arrest in mitosis. In general, 4- chlorochablastine and 4-chlorochacristine caused these effects at concentrations higher than those needed for vinblastine, vincristine, and vinorelbine, but the potency was approximately in the range of vinflunine. Taken together, the results show first indications that these novel vinca alkaloids might be effective and that they warrant further analysis.

Enantioselective synthesis of Iboga alkaloids and vinblastine via rearrangements of quaternary ammoniums.

An efficient and novel strategy for the enantioselective syntheses of various iboga alkaloids has been developed. The salient features include a gold-catalyzed oxidation of a terminal alkyne followed by cyclization, a Stevens rearrangement and a tandem sequence that combines the gold-catalyzed oxidation, cyclization and [1,2]-shift. The catharanthine analogs provided by our approach were further converted to the vinca alkaloid vinblastine and its analogs, which confirmed the remarkable sensitivity of the cytotoxicity to the C20' substituent of vinblastine.

Drug-Resistant Urothelial Cancer Cell Lines Display Diverse Sensitivity Profiles to Potential Second-Line Therapeutics

Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUPrGEMCI20), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer.

ChemInform Abstract: Total Synthesis of Vinblastine, Related Natural Products, and Key Analogues and Development of Inspired Methodology Suitable for the Systematic Study of Their Structure‐Function Properties

AbstractReview: 51 refs.

Total synthesis of vinblastine, related natural products, and key analogues and development of inspired methodology suitable for the systematic study of their structure-function properties.

ConspectusBiologically active natural products composed of fascinatingly complex structures are often regarded as not amenable to traditional systematic structure–function studies enlisted in medicinal chemistry for the optimization of their properties beyond what might be accomplished by semisynthetic modification. Herein, we summarize our recent studies on the Vinca alkaloids vinblastine and vincristine, often considered as prototypical members of such natural products, that not only inspired the development of powerful new synthetic methodology designed to expedite their total synthesis but have subsequently led to the discovery of several distinct classes of new, more potent, and previously inaccessible analogues.With use of the newly developed methodology and in addition to ongoing efforts to systematically define the importance of each embedded structural feature of vinblastine, two classes of analogues already have been discovered that enhance the potency of the natural products >10-fold. In one instance, remarkable progress has also been made on the refractory problem of reducing Pgp transport responsible for clinical resistance with a series of derivatives made accessible only using the newly developed synthetic methodology. Unlike the removal of vinblastine structural features or substituents, which typically has a detrimental impact, the additions of new structural features have been found that can enhance target tubulin binding affinity and functional activity while simultaneously disrupting Pgp binding, transport, and functional resistance. Already analogues are in hand that are deserving of full preclinical development, and it is a tribute to the advances in organic synthesis that they are readily accessible even on a natural product of a complexity once thought refractory to such an approach.

Combined Therapeutic Effects of Vinblastine and Astragalus Saponins in Human Colon Cancer Cells and Tumor Xenograft via Inhibition of Tumor Growth and Proangiogenic Factors

Our previous study had demonstrated that Astragalus saponins (AST) could reduce the side effects of orthodox chemotherapeutic drugs, while concurrently promote antitumor activity. In the present study, we attempted to investigate the potential synergistic anticarcinogenic effects of AST and a vinca alkaloid vinblastine (VBL). Reduced expression of key proangiogenic and metastatic factors including VEGF, bFGF, metalloproteinase (MMP)-2, and MMP-9 was detected in VBL-treated colon cancer cells, with further downregulation by combined VBL/AST treatment. Subsequently, VBL or AST decreased LoVo cell invasiveness, with further reduction when the drugs were cotreated. Significant growth inhibition and cell cycle arrest at G2/M phase were achieved by either drug treatment with apparent synergistic effects. VBL-induced apoptosis was confirmed but found to be unrelated to induction of the novel apoptotic protein NSAID-activated gene 1. In vivo study in tumor xenograft indicates that combined VBL/AST treatment resulted in sustained regression of tumor growth, with attenuation of the neutropenic and anemic effects of VBL. In addition, downregulation of proangiogenic and proliferative factors was also visualized, with boosting effect by combined drug treatment. These findings have provided evidence that AST combined with adjuvant chemotherapeutics like VBL could alleviate cancer development through diversified modes of action, including the regulation of angiogenesis.

Abstract 4493: Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors.

Abstract Eribulin (E7389) is an antimitotic drug that inhibits microtubule assembly in vitro and in vivo by binding to tubulin. Several studies have been performed on eribulin mechanism of action; however, the mode of interaction with soluble tubulin (αβ-heterodimer) and binding site differences with vinca alkaloids (vinblastine and vincristine) and paclitaxel have not been well characterized. To understand binding differences, we performed an in vitro conformational change assays and established a surface plasmon resonance (SPR)-based direct binding assay using chemical probes and soluble tubulin. Unlike vinca alkaloids and paclitaxel, binding of eribulin with tubulin has little or no effect on conformational change of tubulin which is consistent with previous observations in the following three independent assays: 1) limited proteolysis pattern, 2) intrinsic tryptophan fluorescence and 3) exposure of hydrophobic area on the tubulin molecule. We therefore examined whether eribulin binds to the vinca or paclitaxel site of tubulin in competitive inhibition assay on SPR. Vinca alkaloids and paclitaxel failed to inhibit the binding of tubulin to immobilized eribulin chemical probe. Similarly, eribulin and paclitaxel did not inhibit the binding of tubulin to immobilized vinblastine chemical probe. Thus, the eribulin binding site on tubulin is different from that of vinca alkaloids and paclitaxel. In addition, we further investigated the binding affinities and characteristics of interaction of eribulin with GTP- or GDP-forms of tubulin, respectively. Eribulin chemical probe binds both forms of tubulin specifically with significantly higher affinity (∼40 nM) as compared with vinblastine chemical probe (∼1 μM). Interestingly, we found that eribulin chemical probe preferentially recognizes GTP- over GDP-tubulin and dissociation of GTP-form from eribulin chemical probe was slower than that of GDP-form of tubulin. In contrast, vinblastine chemical probe did not show significant difference in binding to the two nucleotide states of tubulin. This indicates that eribulin distinguishes different nucleotide state of tubulin and binds to GTP-tubulin more tightly. These characteristic features of eribulin differ from those of vinca alkaloids and paclitaxel. Overall, our results suggest that eribulin exerts its unique antimitotic activity by inhibiting microtubule assembly by binding with high affinity at a novel tubulin site at the plus-end of the growing microtubule. This may potentially relate to the mechanism of eribulin clinical efficacy and diminished neuronal toxicity. Citation Format: Kishan Lal Agarwala, Kenji Kubara, Koji Sagane, Boris M. Seletsky, Bruce A. Littlefield. Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2013-4493

Anticancer compounds from plants.

Cancer is a leading cause of death all over the world and represents a major public health burden. Natural plant products have been historically used for the treatment of various diseases. The earnest search for plant-derived anticancer agents began in the 1950s with the discovery and development of the vinca alkaloids–vinblastine and vincristine, and with the isolation of the cytotoxic podophyllotoxins (Cragg and Newman, 2005). A good number of the current– day commercially approved anticancer drugs as well as the natural product-derived compounds in various stages of clinical development as anticancer agents originate from plants. Natural products still serve as an excellent source for the discovery and development of modern drugs for cancer treatment. The present work reviews the most recent information in the field of natural anticancer substances, and in this regard we have summarized the key information published in 2012 on plant derived anticancer compounds (Table 1).

Characterization of functional activity of ABCB1 and ABCC1 proteins in eggs and embryonic cells of the sea urchin Echinometra lucunter

ABC transporter (ATP-binding-cassette transporter) proteins have been strongly associated with the phenomenon of multidrug resistance in cancer cells. Furthermore, their physiological expression has been studied in many organisms, including bacteria, fungi, plants and vertebrate or invertebrate animals. Their widespread expression through the evolution demonstrates their relevance to the survival of living things. In the present study, we characterized the functional activity of ABCB1 and ABCC1 proteins in gametes and embryonic cells of the sea urchin Echinometra lucunter. The ABC transporter proteins' functional activity was up-regulated post-fertilization. Eggs and spermatozoa of E. lucunter accumulated more C-AM (calcein acetoxymethyl ester), a fluorescent substrate of ABCB1 and ABCC1 proteins, than embryonic cells. Verapamil, reversin 205 and indomethacin were able to increase C-AM influx in eggs and embryos. However, verapamil and reversin 205 were more efficient than indomethacin, suggesting a predominance of ABCB1 protein over ABCC1 protein activity. Multidrug resistance modulating agents, at the concentration range that inhibited ABC transporter proteins, did not block the embryonic development until blastula stage. However, inhibition of ABCB1-mediated efflux by reversin 205 circumvented resistance of embryos to the antimitotic vinca alkaloid vinblastine. Embryonic development was more efficiently blocked when colchicine was previously added to eggs than to embryos 5 min after fertilization. This set of results suggests that these proteins act as a fundamental biochemical barrier conferring a protective physiological role against toxic xenobiotics in E. lucunter embryos.

Identification of the Major Phosphorylation Site in Bcl-xL Induced by Microtubule Inhibitors and Analysis of Its Functional Significance

Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 --> Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 --> Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.

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.

Cytotoxic chemotherapy in advanced non-small cell lung cancer

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Flavins and flavoenzymes in diagnosis and therapy.

AbstractRiboflavin is a vitamin but not at all harmless. It is therefore not surprising that riboflavin and the riboflavinogenic coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play a role in many fields of practical medicine. Here we report on new developments in medical flavinology focusing on the assessment of riboflavin status, medical interventions based on flavins, and current medical aspects concerning the enzymes glutathione reductase (GR) and thioredoxin reductase (TrxR) as representatives of a large family of homodimeric flavoproteins. For systematic and more specific information—particularly on the clinical aspects—we should like to refer the reader to comprehensive reviews (1–4). This article cannot deal with one specific method. However, methodological details of special medical interest are included. We will keep recalling a simple rule that is often broken in practical medicine and in medical research: Drugs, B2 and light? The treatment can’t be right. It implies that flavins are readily photodegraded, and that a whole range of drugs (5,6) but also biological macromolecules (3,7,8) are subject to inactivating modification in riboflavin-dependent photoreactions. As exemplified for the cytostatic Vinca alkaloids vinblastine and vincristine as well as for the synthetic drug vindesine precautions have to be taken to prevent this side effect of riboflavin application (6).KeywordsGlutathione ReductaseFlavin Adenine DinucleotideFlavin Adenine DinucleotideCytochrome P450 ReductaseRiboflavin DeficiencyThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Enhancement of chemotherapeutic drug toxicity to human tumour cells in vitro by a subset of non-steroidal anti-inflammatory drugs (NSAIDs)

The effect on cytotoxicity of combining a range of clinically important non-steroidal anti-inflammatory drugs (NSAIDs) with a variety of chemotherapeutic drugs was examined in the human lung cancer cell lines DLKP, A549, COR L23P and COR L23R and in a human leukaemia line HL60/ADR. A specific group of NSAIDs (indomethacin, sulindac, tolmetin, acemetacin, zomepirac and mefenamic acid) all at non-toxic levels, significantly increased the cytotoxicity of the anthracyclines (doxorubicin, daunorubicin and epirubicin), as well as teniposide, VP-16 and vincristine, but not the other vinca alkaloids vinblastine and vinorelbine. A substantial number of other anticancer drugs, including methotrexate, 5-fluorouracil, cytarabine, hydroxyurea, chlorambucil, cyclophosphamide, cisplatin, carboplatin, mitoxantrone, actinomycin D, bleomycin, paclitaxel and camptothecin, were also tested, but displayed no synergy in combination with the NSAIDs. The synergistic effect was concentration dependent. The effect appears to be independent of the cyclo-oxygenase inhibitory ability of the NSAIDs, as (i) the synergistic combination could not be reversed by the addition of prostaglandins D 2 or E 2; (ii) sulindac sulphone, a metabolite of sulindac that does not inhibit the cyclooxygenase enzyme, was positive in the combination assay: and (iii) many NSAIDs known to be cyclo-oxygenase inhibitors, e.g. meclofenamic acid, diclofenac, naproxen, fenoprofen, phenylbutazone, flufenamic acid, flurbiprofen, ibuprofen and ketoprofen, were inactive in the combination assay. The enhancement of cytotoxicity was observed in a range of drug sensitive tumour cell lines, but did not occur in P-170-overexpressing multidrug resistant cell lines. However, in the HL60/ADR and COR L23R cell lines, in which multidrug resistance is due to overexpression of the multidrug resistance-associated protein MRP, a significant increase in cytotoxicity was observed in the presence of the active NSAIDs. Subsequent Western blot analysis of the drug sensitive parental cell lines, DLKP and A549, revealed that they also expressed MRP and reverse-transcription–polymerase chain reaction studies demonstrated that mRNA for MRP was present in both cell lines. It was found that the positive NSAIDs were among the more potent inhibitors of [ 3H]-LTC 4 transport into inside-out plasma membrane vesicles prepared from MRP-expressing cells, of doxorubicin efflux from preloaded cells and of glutathione-S-transferase activity. The NSAIDs did not enhance cellular sensitivity to radiation. The combination of specific NSAIDs with anticancer drugs reported here may have potential clinical applications, especially in the circumvention of MRP-mediated multidrug resistance.

Influence of vinblastine on DNA parameters and multidrug resistance in renal cell carcinoma in vitro.

The purpose of this study was to investigate how the vinca alkaloid vinblastine influences DNA parameters and the mechanisms of multidrug resistance in renal cell carcinoma. After exposing cell cultures of human renal carcinoma to progressively increasing concentrations of vinblastine the cell lines were examined by flow cytometric DNA analysis to assess the S-phase and G2/M-phase fraction and by a modified MTT assay. It was shown that the exposed cells became P-glycoprotein-positive by staining the cells with a monoclonal antibody (JSB-1). The flow cytometric analysis revealed, with prolonged vinblastine exposure, correlated increases in the S-phase and G2/M-phase fractions (P = 0.0001). When vinblastine-free medium was used for culturing, the changed DNA characteristics returned to their original values. Comparing the DNA parameters with the IC50 (concentration when cell growth is inhibited by 50%) we found a strong correlation between these parameters (P = 0.0001). In conclusion, DNA analysis of long-term vinblastine exposure may provide insight into events leading to multidrug resistance. Furthermore, analysis of the DNA profile might also be an important investigation before planning therapy with vinblastine for renal cell carcinoma.

New Chemotherapeutic Agents for Non-small Cell Lung Cancer

The identification of new chemotherapeutic agents for the treatment of non-small cell lung cancer should proceed in a structured, logical fashion. Agents should be evaluated on the basis of multiple objective and subjective end points. A 15% or greater major objective response rate, demonstrated in multiple single-agent phase II trials, is considered the lower limit for an agent to be deemed clinically active in this disease. A number of drugs previously have been identified in this category, including cisplatin, ifosfamide, mitomycin, paclitaxel, and the vinca alkaloids vinblastine and vindesine. Most of these conventional agents have been explored alone, in a variety of doses and schedules, and in combination. In the last several years clinical development has produced new agents, including chloroquinoxaline sulfonamide, docetaxel, edatrexate, gemcitabine, irinotecan, topotecan, and vinorelbine, which hold promise for more successful treatment of this lethal disease.