Artificial Metalloenzymes with the Neocarzinostatin Scaffold: Toward a Biocatalyst for the Diels-Alder Reaction.

A copper(II) cofactor coupled to a testosterone anchor, copper(II)-(5-(Piperazin-1-yl)-1,10-phenanthroline)testosterone-17-hemisuccinamide (10) was synthesized and associated with a neocarzinostatin variant, NCS-3.24 (KD =3 µm), thus generating a new artificial metalloenzyme by following a "Trojan horse" strategy. Interestingly, the artificial enzyme was able to efficiently catalyze the Diels-Alder cyclization reaction of cyclopentadiene (1) with 2-azachalcone (2). In comparison with what was observed with cofactor 10 alone, the artificial enzymes favored formation of the exo products (endo/exo ratios of 84:16 and 62:38, respectively, after 12 h). Molecular modeling studies assigned the synergy between the copper complex and the testosterone (KD =13 µm) moieties in the binding of 10 to good van der Waals complementarity. Moreover, by pushing the modeling exercise to its limits, we hypothesize on the molecular grounds that are responsible for the observed selectivity.

Neocarzinostatin-based hybrid biocatalysts with a RNase like activity.

A new zinc(II)-cofactor coupled to a testosterone anchor, zinc(II)-N,N-bis(2-pyridylmethyl)-1,3-diamino-propa-2-ol-N'(17'-succinimidyltestosterone) (Zn-Testo-BisPyPol) 1-Zn has been synthesized and fully characterized. It has been further associated with a neocarzinostatin variant, NCS-3.24, to generate a new artificial metalloenzyme following the so-called 'Trojan horse' strategy. This new 1-Zn-NCS-3.24 biocatalyst showed an interesting catalytic activity as it was found able to catalyze the hydrolysis of the RNA model HPNP with a good catalytic efficiency (kcat/KM=13.6M(-1)s(-1) at pH 7) that places it among the best artificial catalysts for this reaction. Molecular modeling studies showed that a synergy between the binding of the steroid moiety and that of the BisPyPol into the protein binding site can explain the experimental results, indicating a better affinity of 1-Zn for the NCS-3.24 variant than testosterone and testosterone-hemisuccinate themselves. They also show that the artificial cofactor entirely fills the cavity, the testosterone part of 1-Zn being bound to one the two subdomains of the protein providing with good complementarities whereas its metal ion remains widely exposed to the solvent which made it a valuable tool for the catalysis of hydrolysis reactions, such as that of HPNP. Some possible improvements in the 'Trojan horse' strategy for obtaining better catalysts of selective reactions will be further studied.

Thiols screened by the neocarzinostatin protein for preserving or detoxifying its bound enediyne antibiotic.

Neocarzinostatin is an antibiotic chromoprotein produced by Streptomyces carzinostaticus. Its enediyne-containing chromophore exhibits high DNA cleavage activity and belongs to one of the most potent categories of antitumor agents. The labile chromophore is readily inactivated by environmental thiols including the most abundant glutathione. How the microorganism preserves the secreted antibiotic and at the same time is immune to its toxicity are of interest. Site-directed mutagenesis studies of the neocarzinostatin protein have shown that residues D33 and D99 play primary and secondary roles, respectively, in preserving neocarzinostatin from acidic glutathione whereas D79 and other residues around the opening of the binding cleft have an insignificant effect. Biothiol analyses revealed that cells of S. carzinostaticus produced no glutathione, but instead neutral mycothiol, which is known to serve functions analogous to glutathione. Mycothiol was the only neutral-charged thiol produced by the organism; all other identified biothiols carried at least partial negative charges. When the bacteria were cultured under conditions that stimulated the biosynthesis of neocarzinostatin, the yield of mycothiol increased significantly, which suggests mycothiol-dependent cellular detoxification. Treating neocarzinostatin samples with the cell extract that retained active sulfhydryls led to efficient drug inactivation, which indicates that mycothiol is allowed to approach the protein-bound chromophore. The anionic side-chains of D33 and D99 in the neocarzinostatin protein played two critical roles in a single thiol-screening operation: Preserving the antibiotic for defense and survival by rejecting the ubiquitous glutathione through charge-charge repulsion in the outer-cell environment and detoxifying the toxin in the inner-cell body for self-resistance by accepting the cell-produced neutral mycothiol.

Lipid bilayer-assisted release of an enediyne antibiotic from neocarzinostatin chromoprotein.

The nine-membered enediyne class has drawn extensive interest because of extremely high antitumor potency and intricate interactions with its carrier protein. While the drug-induced DNA cleavage reactions have been mostly elucidated, the critical release-transport process of the labile enediyne molecule in cellular environment remained obscure. Using neocarzinostatin chromoprotein as a model, we demonstrated a lipid bilayer-assisted release mechanism. The in vitro enediyne release rate under aqueous conditions was found to be too slow to account for its efficient DNA cleavage action. Via the presence of lipid bilayers, chaotropic agents, or organic solvents, we found the release was substantially enhanced. The increased rate was linearly dependent on the lipid bilayer concentration and the dielectric value of the binary organic solvent mixtures. While lipid bilayers provided a low surrounding dielectricity to assist in drug release, there were no major conformational changes in the apo and holo forms of the carrier protein. In addition, the lifespan of the released enediyne chromophore was markedly extended through partitioning of the chromophore in the hydrophobic bilayer phase, and the lipid bilayer-stabilized enediyne chromophore significantly enhanced DNA cleavage in vitro. Collectively, we depicted how a lipid bilayer membrane efficiently enhanced dissociation of the enediyne chromophore through a hydrophobic sensing release mechanism and then acted as a protector of the released enediyne molecule until its delivery to the target DNA. The proposed membrane-assisted antibiotic release-transport model might signify a new dimension to our understanding of the modus operandi of the antitumor enediyne drugs.

Molecular dynamics simulations investigation of neocarzinostatin chromophore-releasing pathways from the holo-NCS protein.

The enediyne ring chromophore with strong DNA cleavage activity of neocarzinostatin is labile and therefore stabilization by forming the complex (carrying protein+chromophore: holo-NCS). Holo-NCS has gained much attention in clinical use as well as for drug delivery systems, but the chromophore-releasing mechanism to trigger binding to the target DNA with high affinity and producing DNA damage remain unclear. Three possible pathways were initially determined by conventional MD, essential dynamics and essential dynamics sampling. One of the paths runs along the naphthoate moiety; another runs along the amino sugar moiety; the third along the enediyne ring. Further, calculated forces and time by FPMD (force-probe molecular dynamics) suggest that the opening of the naphthoate moiety is most favorable pathway and Leu45, Phe76 and Phe78 all are key residues for chromophore release. In addition, conformational analyses indicate that the chromophore release is only local motions for the protein.

Insight into the strong inhibitory action of salt on activity of neocarzinostatin.

Enediyne anticancer drugs belong to one of the most potent category in inducing DNA damage. We report 85+/-5% inhibition on activity of neocarzinostatin by salt. As high sodium ion concentration is a known tumor cell feature, we explored the dynamic mechanism of inhibition. Using various analytical tools, we examined parameters involved in the four consecutive steps of the drug action, namely, drug releasing from carrier protein, drug-DNA binding, drug activating, and DNA damaging. Neither protein stability, nor drug release rate, was altered by salt. The salt inhibition level was similar in between the protein-bound and unbound enediyne chromophore. Salt did not quench the thiol-induced drug activation. The inhibition was independent of DNA lesion types and irrelevant with thiol structures. Collectively, no salt interaction was found in the releasing, activating, and DNA damaging step of the drug action. However, binding with DNA decreased linearly with salt and corresponded well with the salt-induced inhibition on the drug activity. Salt interference on the affinity of DNA binding was the main and sole cause of the severe salt inhibition. The inhibition factor should be carefully considered for all agents with similar DNA binding mode.

Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin.

The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

Disulfide bond substitution by directed evolution in an engineered binding protein.

Breaking ties: The antitumour protein, neocarzinostatin (NCS), is one of the few drug-carrying proteins used in human therapeutics. However, the presence of disulfide bonds limits this protein's potential development for many applications. This study describes a generic directed-evolution approach starting from NCS-3.24 (shown in the figure complexed with two testosterone molecules) to engineer stable disulfide-free NCS variants suitable for a variety of purposes, including intracellular applications.The chromoprotein neocarzinostatin (NCS) has been intensively studied for its antitumour properties. It has recently been redesigned as a potential drug-carrying scaffold. A potential limit of this protein scaffold, especially for intracellular applications, is the presence of disulfide bonds. The objective of this work was to create a disulfide-free NCS-derived scaffold. A generic targeted approach was developed by using directed evolution methods. As a starting point we used a previously engineered NCS variant in which a hapten binding site had been created. A library was then generated in which cysteine Cys88 and Cys93 and neighbouring residues were randomly substituted. Variants that preserved the hapten binding function were selected by phage display and further screened by colony filtration methods. Several sequences with common features emerged from this process. The corresponding proteins were expressed, purified and their biophysical properties characterised. How these selected sequences rescued folding ability and stability of the disulfide-free protein was carefully examined by using calorimetry and the results were interpreted with molecular simulation techniques.

DNA conformation-induced activation of an enediyne for site-specific cleavage.

Neocarzinostatin chromophore (NCS chrom) was found to induce site-specific cleavage at the 3' side of a bulge in single-stranded DNA in the absence of thiol. This reaction involved the oxidative formation of a DNA fragment with a nucleoside 5'-aldehyde at its 5' terminus and generated an ultraviolet light-absorbing and fluorescent species of post-activated drug containing tritium abstracted from the carbon at the 5' position of the target nucleotide. The DNAs containing point mutations that disrupt the bulge were not cleavage substrates and did not generate this drug product. Thus, DNA is an active participant in its own destruction, and NCS chrom may be useful as a probe for bulged structures in nucleic acids.

Solution structure of a two-base DNA bulge complexed with an enediyne cleaving analog.

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore in a base-catalyzed, radical-mediated reaction. The solution structure of the complex between an analog of the bulge-specific cleaving species and an oligodeoxynucleotide containing a two-base bulge was elucidated by nuclear magnetic resonance. An unusual binding mode involves major groove recognition by the drug carbohydrate unit and tight fitting of the wedge-shaped drug in the triangular prism pocket formed by the two looped-out bulge bases and the neighboring base pairs. The two drug rings mimic helical DNA bases, complementing the bent DNA structure. The putative abstracting drug radical is 2.2 +/- 0.1 angstroms from the pro-S H5' of the target bulge nucleotide. This structure clarifies the mechanism of bulge recognition and cleavage by a drug and provides insight into the design of bulge-specific nucleic acid binding molecules.

Is association of labile enediyne chromophore a mutually assured protection for carrier protein?

Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein-chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the protein-bound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability.

Selective elimination of anti-DNA antibody-producing cells by antiidiotypic antibody conjugated with neocarzinostatin.

A new strategy was shown for the manipulation of autoantibody production in humans. Antiidiotypic antibody to human anti-DNA autoantibody was conjugated with neocarzinostatin (NCS), a cytotoxic agent, by using N-succimidyl 3-(2-pyridyldithio) propionate as a coupling agent. Human B cell clones, which produce anti-DNA autoantibodies, were killed by in vitro treatment with antiidiotype (Id)-NCS conjugates, while clones expressing an Id with irrelevant specificity were unaffected. These results indicate that treatment with anti-Id-NCS conjugates can act as a potent and specific means of generating immunosuppression of autoantibody production. This approach will have a significant advantage in aborting clones that are not effectively suppressed for the autoantibodies by anti-Id antibodies alone, and will result in a potential therapeutic treatment for systemic lupus erythematosus.

Structures of in vitro evolved binding sites on neocarzinostatin scaffold reveal unanticipated evolutionary pathways.

We have recently applied in vitro evolution methods to create in Neocarzinostatin a new binding site for a target molecule unrelated to its natural ligand. The main objective of this work was to solve the structure of some of the selected binders in complex with the target molecule: testosterone. Three proteins (1a.15, 3.24 and 4.1) were chosen as representative members of sequence families that came out of the selection process within different randomization schemes. In order to evaluate ligand-induced conformational adaptation, we also determined the structure of one of the proteins (3.24) in the free and complexed forms. Surprisingly, all these mutants bind not one but two molecules of testosterone in two very different ways. The 3.24 structure revealed that the protein spontaneously evolved in the system to bind two ligand molecules in one single binding crevice. These two binding sites are formed by substituted as well as by non-variable side-chains. The comparison with the free structure shows that only limited structural changes are observed upon ligand binding. The X-ray structures of the complex formed by 1a.15 and 4.1 Neocarzinostatin mutants revealed that the two variants form very similar dimers. These dimers were observed neither for the uncomplexed variants nor for wild-type Neocarzinostatin but were shown here to be induced by ligand binding. Comparison of the three complexed forms clearly suggests that these unanticipated structural responses resulted from the molecular arrangement used for the selection experiments.

Mechanisms of DNA cleavage by copper complexes of 3-clip-phen and of its conjugate with a distamycin analogue.

Mechanisms of DNA oxidation by copper complexes of 3-Clip-Phen and its conjugate with a distamycin analogue, in the presence of a reductant and air, were studied. Characterisation of the production of 5-methylenefuranone (5-MF) and furfural, associated with the release of nucleobases, indicated that these copper complexes oxidised the C1' and C5' positions of 2-deoxyribose, respectively, which are accessible from the DNA minor groove. Oxidation at C1' was the major degradation route. Digestion of DNA oxidation products by P1 nuclease and bacterial alkaline phosphatase allowed characterisation of glycolic acid residues, indicating that these copper complexes also induced C4' oxidation. However, this pathway was not associated with base propenal release. The ability of the copper complex of the 3-Clip-Phen conjugate with the distamycin analogue to produce sequence-selective DNA cleavage allowed confirmation of these mechanisms of DNA oxidation by PAGE. Comparison of DNA cleavage activity showed that conjugation of 3-Clip-Phen with a DNA minor groove binder, like the distamycin analogue, decreased both its ability to perform C1' oxidation as well as the initial rate of the reaction, but this conjugate is still active after 5 h at 37 degrees C, making it an efficient DNA cleaver.

Facilitated internalization of neocarzinostatin and its lipophilic polymer conjugate, SMANCS, into cytosol in acidic pH.

The effects of environmental pH on the binding and cytotoxicity of the antitumor proteins neocarzinostatin (NCS) and SMANCS [copoly(styrene-maleic acid)-conjugated NCS] to cultured cells were studied by using their fluorescent-labeled derivatives (F-drugs). At 37 degrees C the binding of these drugs to HeLa cells was pH dependent: The amount of cell-bound drugs increased with an increase in the acidity of the medium. The pH-dependent change in the binding of the drugs was not as evident at 0 degree C. The cytotoxic action of these drugs was much more rapid at acidic pH compared with that at neutral or slightly alkaline pH. Furthermore, F-drugs could be utilized to probe the microenvironmental pH in Meth-A cells, in which the drug was located by the ratio of fluorescent intensities at 450 and 490 nm. The environment of the cell-bound F-drugs became acidic with incubation time at 37 degrees C but not at 0 degree C. Inasmuch as these drugs directly attack DNA, these results suggest that NCS and SMANCS are translocated across the membrane of acidic vesicles into the cytosol after endocytotic uptake. This hypothesis is also supported by the finding that NH4Cl and chloroquine protected HeLa cells against the cytotoxicity of the drugs. Data also showed that the hydrophobic polyanion conjugate SMANCS had a much greater cell binding (10 times) and more rapid internalization compared with NCS. Taken together, our results show that acidic pH of tumor tissue is preferable for effective binding and internalization into cytosol for NCS and SMANCS.

Binding to and internalization by cultured cells of neocarzinostatin and enhancement of its actions by conjugation with lipophilic styrene-maleic acid copolymer.

The binding of a copoly(styrene-maleic acid)-conjugated neocarzinostatin (NCS) designated as smancs (Mr 16,000), and parental NCS (Mr 12,000) to cultured cells was investigated. These drugs were labeled with fluorescein isothiocyanate which retained biological activity and were used for binding studies. The binding of these drugs to HeLa cells was dependent on time and temperature, with 2 times more drug being bound at 37 degrees C than at 0 degree C. In the presence of a 100-fold molar excess of unlabeled NCS, the binding of smancs or NCS to HeLa cells was inhibited similarly. Therefore, it was suggested that smancs binds to NCS-binding sites (receptor) of the cell surface. However, the amount of cell-bound smancs was increased about 20-fold compared with that of NCS. Scatchard plot analyses of the binding of these drugs to HeLa and WISH cells indicated that this increase was due to alterations in affinity resulting from polymer conjugation of the drug to receptor rather than to an increase in the number of drug-binding sites at the cell surface. Furthermore, when the cytotoxicity of these drugs to HeLa cells was compared, smancs needed only 5 min to achieve 50% inhibition of the control. In contrast, the same dose level of NCS required more than 90 min to achieve the same toxic effect. More rapid internalization of smancs than NCS was also elucidated under fluorescence microscopy at 37 degrees C. There was no intracellular incorporation of these drugs below 20 degrees C. These results indicated that an increased lipophilicity of smancs appears to be responsible for its increased cell surface affinity, internalization rate, and toxic effect. Concordant to this interpretation was that among various copoly(styrene-maleic acid) esters, a more hydrophobic derivative showed more internalization (butyl greater than ethyl greater than carboxylate). These results suggest the possibility of utilizing protein tailoring to augment the subcellular activity of functional proteins.

Glutathione dependence of neocarzinostatin cytotoxicity and mutagenicity in Chinese hamster V-79 cells.

Neocarzinostatin (NCS) is mutagenic in bacteria, yeast, fungi, and mammalian cells. In cell-free systems, DNA strand breakage induced by NCS requires a reducing agent like 2-mercaptoethanol, unless very high (greater than 100 micrograms/ml) concentrations of NCS are used. In this study, we have investigated the role of the sulfhydryl compound glutathione (GSH), which is usually the most common intracellular thiol, in the bioactivation of NCS to a toxic and mutagenic species. Chinese hamster V79 cells were pretreated with one of two GSH depleting agents, buthionine sulfoximine or diethyl maleate. These agents deplete GSH via different mechanisms, but both will lower GSH levels within the cell to less than 5% of control (untreated) values. GSH-depleted cells and control cells were then exposed to NCS concentrations of 0.5-2.5 micrograms/ml for 1 h, assayed for survival, and plated for expression of hypoxanthine-guanine phosphoribosyltransferase-negative (HGPRT-) mutants. After an expression period of 7 days, during which the cultures were subcultured twice, HGPRT- mutants were selected by plating in hypoxanthine-free medium containing 5 micrograms of 6-thioguanine per ml, at a density of 2 X 10(5) cells per 100 mm dish. NCS alone decreased the surviving fraction to about 1% at 2.5 micrograms/ml and produced dose-related increases in HGPRT-mutants that reached greater than 10 times the spontaneous mutation frequency at 2.5 micrograms NCS per ml. In GSH-depleted cells, however, NCS was only mildly cytotoxic (60-80% surviving fraction) and did not produce dose-related increases in HGPRT- mutants over cells treated only with diethyl maleate or buthionine sulfoximine. Thus, GSH appears to be the main reducing agent for the bioactivation of NCS to a toxic and mutagenic species in Chinese hamster V79 cells.

A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs.

We previously found that a polymer conjugated to the anticancer protein neocarzinostatin, named smancs, accumulated more in tumor tissues than did neocarzinostatin. To determine the general mechanism of this tumoritropic accumulation of smancs and other proteins, we used radioactive (51Cr-labeled) proteins of various molecular sizes (Mr 12,000 to 160,000) and other properties. In addition, we used dye-complexed serum albumin to visualize the accumulation in tumors of tumor-bearing mice. Many proteins progressively accumulated in the tumor tissues of these mice, and a ratio of the protein concentration in the tumor to that in the blood of 5 was obtained within 19 to 72 h. A large protein like immunoglobulin G required a longer time to reach this value of 5. The protein concentration ratio in the tumor to that in the blood of neither 1 nor 5 was achieved with neocarzinostatin, a representative of a small protein (Mr 12,000) in all time. We speculate that the tumoritropic accumulation of these proteins resulted because of the hypervasculature, an enhanced permeability to even macromolecules, and little recovery through either blood vessels or lymphatic vessels. This accumulation of macromolecules in the tumor was also found after i.v. injection of an albumin-dye complex (Mr 69,000), as well as after injection into normal and tumor tissues. The complex was retained only by tumor tissue for prolonged periods. There was little lymphatic recovery of macromolecules from tumor tissue. The present finding is of potential value in macromolecular tumor therapeutics and diagnosis.

In vitro inhibition of human leukemic cells (CCRF-CEM) by agarose-immobilized neocarzinostatin.

Neocarzinostatin (NCS) is an acidic protein (molecular weight, 10,700) isolated from Streptomyces carzinostaticus that has antitumor activity both in model rodent systems and in humans. In vitro it inhibits the growth of a human lymphoblastic leukemic cell line (CCRF-CEM) at a very low concentration (the amount of drug that causes a 50% inhibition of growth compared to control cultures as extrapolated from a dose-response curve (ID50), 2.4 X 10(-9) M). We covalently coupled NCS to the N-hydroxysuccinimide ester of agarose and obtained a product that, by a variety of biochemical and immunological criteria, has been demonstrated to be devoid of any free or loosely bound NCS. Agarose-bound NCS, which is unable to enter cells because of its size, retains a significant amount of inhibitory activity (ID50, 6 to 15 X 10(-9) M) and is also capable of inhibiting tritiated deoxythymidine incorporation into CCRF-CEM cells. Since agarose-bound NCS cannot enter mammalian cells, the above findings indicate that NCS is able to exert its toxic effects by binding to or reacting with receptors on the cell membrane.

Comparison of the cytotoxic effects of the high- and low-molecular-weight anticancer agents on multidrug-resistant Chinese hamster ovary cells in vitro.

Neocarzinostatin (NCS), styrene-maleic acid copolymer-conjugated neocarzinostatin (SMANCS), and ricin exhibited cytotoxicity against two different types of Chinese hamster ovary cells, parental AUXB1 cells and the multidrug-resistant (MDR) subline CHRC5 cells at the nanomolar range. These doses were much lower than those of the other anticancer drugs tested (micromolar range), even after a short incubation. MDR CHRC5 cells were 20 to 900 times more resistant to Adriamycin, aclacinomycin, vinblastine, and mitomycin C than were AUXB1 cells. However, the resistance of CHRC5 cells to NCS, SMANCS, or ricin was relatively low: the 50% colony inhibitory concentration was only 5 to 10 times higher than that for parental AUXB1 cells. CHRC5 cells were not resistant to 5-fluorouracil and cis-diamminedichloroplatinum(II), but the effective doses of these agents to them were 10(3)-10(6) times higher, and longer incubation times were required to produce the same cytotoxicity as NCS and SMANCS. Furthermore, cell-bound NCS, SMANCS, and ricin were not released from AUXB1 or CHRC5 cells during a 120-min incubation, although Adriamycin was excreted very rapidly from CHRC5 cells after binding and internalization. These results strongly suggest that NCS, SMANCS, and ricin, which are internalized into cells by endocytosis, were not excreted from the cells by active efflux and exhibited a pronounced anticancer effect against MDR cells.