Cytotoxicity toxins

Following the first report of tumor promotion by okadaic acid, additional tumor promoters of the okadaic acid activity class have been identified, e.g., microcystin [513,514], and calyculin derivatives, Fig. (63) reported in marine sponges such as Discodermia calyx [515] and Theonella swinhoei [516] as potent inhibitors of tumor cell proliferation. A two-sponge association, Poecillastra sp. and Jaspis sp., yielded cytotoxic toxins which exhibited selective activity against several tumoral cell lines [517],... 

Contaminants pose a special danger to human or animal health. In the case of human health, these include, but are not limited to, penicillin, cephalosporins, cytotoxics, toxins, and infectious agents and... 

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

Many of the toxins obtained from coelenterates and echinoderms, because of their hemolytic or cytotoxic actions, are assumed to have a general disruptive action on cell membranes. However, since many of these toxins are capable of forming pores or channels in the plasma membrane of cells, their cytolytic actions may be a result of this highly selective action. On the other hand, the saponins from starfish and sea cucumbers have a direct lytic action as a result of their detergent action on the integrity of cells. 

In oncology, to study the relationship between the normal and the tumour cell, to detect tumour-associated antigens (CEA, carcino-embryonic antigen, and AFP, a-fetoprotein) and subsequently to enable cancer therapy to be monitored, to locate tumour metastases, and to deliver cytotoxic drugs, toxins, radionuclides, or liposomes to tumour cells. 

Examples of inhibitors of chromatin function derived from flowering plants (Fig. 80) are etoposide (lignan) and alkaloids camptothecin, Vinca alkaloids, and 7 epitaxol. The rhizome of Podophyllum peltatum L. (May apple, Berberidaceae) has been used to remove warts and to relieve the bowels from costiveness since very early times. It contains podophyllo-toxin, a cytotoxic lignan from which etoposide (Vepesid ), which is used to treat lung cancer, lymphomas, and leukemias on account of its ability to inhibit the activity of... 

Vaccination to induce an adaptive immune response is expected for a broad range of infectious diseases and cancers. Traditional vaccines are mainly composed of live attenuated viruses, whole inactivated pathogens, or inactivated bacterial toxins. In general, these approaches have been successful for developing vaccines that can induce an immune response based on antigen-specific antibody and cytotoxic T lymphocyte (CTL) responses, which kill host cells infected with intracellular organisms (Fig. 1) [1,2], One of the most important current issues in vaccinology is the need for new adjuvants (immunostimulants) and delivery systems. Many of the vaccines currently in development are based on purified subunits, recombinant... 

Cell cytotoxicity assays with the encapsulated L929 cells were performed. Methanol and CoCl2 solutions were used as toxins. Whereas methanol is a strong... 

Protein toxin possessing cytotoxic properties in vivo... 

Figure 21.1 The basic design of an immunotoxin conjugate consists of an antibody-targeting component crosslinked to a toxin molecule. The complexation typically includes a disulfide bond between the antibody portion and the cytotoxic component of the conjugate to allow release of the toxin intracellularly. In this illustration, an intact A-B toxin protein provides the requisite disulfide, but the linkage also may be designed into the crosslinker itself.

Figure 21.2 Conceptualized construction of an A-B subunit protein toxin (left). The B chain contains a binding region for docking onto cell surfaces, while the A chain contains a catalytic site that produces cytotoxic affects intracellularly. The two subunits are joined by a disulfide bond that is reductively cleaved at the cellular level to allow the A subunit to affect cell death. A molecular model of ricin is on the right.

A-chain immunotoxins, however, may not be quite as cytotoxic as conjugates formed from intact toxin molecules (Manske et al., 1989). In an alternative approach to A chain use, the intact toxin of two-subunit proteins is directly conjugated to a monoclonal without isolation of the A chain. Conjugation of an antibody with intact A-B chain toxins can be done without a cleavable linker, as long as the A chain can still separate from the B chain once it is internalized. Therefore, it is important to avoid intramolecular crosslinking during the conjugation process which can prevent release of the A-B complex. In addition, since the B chain... 

The toxin component of the conjugate must be able to elicit cytotoxicity by ribosomal damage as it could in its native state. This means that the cell penetration and enzymatic properties of the toxin remain unaltered, although an antibody molecule is conjugated to it. 

Figure 21.7 An intact A-B subunit toxin molecule may be activated with 2-iminothiolane with good retention of cytotoxic activity. The thiolated toxin then may be conjugated with SPDP-activated antibody to generate the immunotoxin conjugate through a disulfide bond.

However, since SMCC forms nonreversible thioether linkages with sulfhydryl groups, it only can be used in the preparation of immunotoxins if intact A-B toxins are employed in the conjugate. In such conjugates, the A chain still have the potential for reductive release from the B-chain subunit after cellular docking and internalization. Immunotoxins prepared with A-chain or single-subunit toxins will not display cytotoxicity if crosslinked with SMCC, since the crosslinker does not create cleavable disulfide bonds upon conjugation. 

Figure 21.13 Sulfo-SMCC may be used to activate antibody molecules for coupling to thiolated toxin components. An intact A-B toxin molecule can be modified to contain sulfhydryls by treatment with 2-iminothiolane. Thiolation with this reagent retains the cytotoxic properties of the toxin while generating a sulfhydryl for conjugation. Reaction of the thiolated toxin with the maleimide-activated antibody creates the immunotoxin through thioether bond formation.

The method for the preparation of immunotoxins with SMPB is identical to that used for MBS (above). Since the thioether bonds formed with sulfhydryl-containing molecules are non-cleavable, A-chain isolates or single-chain toxin molecules can not be conjugated with antibodies with retention of cytotoxicity. Only intact A-B toxin molecules may be used with this crosslinker, since the A chain still is capable of being reductively released from the complex. 

Dell Arciprete, L., Colombatti, M., Rappuoli, R., and Tridente, G. (1988) A C terminus cysteine of diphtheria toxin B chain involved in immunotoxin cell penetration and cytotoxicity./. Immunol. 140, 2466-2471. 

Jansen, F.L., Blythman, H.E., Carriere, D., Casellas, P., Diaz, J., Gros, P., Hennequin, J.R., Paolucci, F., Pau, B., Poncelet, P., Richer, G., Salhi, S.L., Vidal, H., and Voisin, G.A. (1980) High specific cytotoxicity of antibody-toxin hybrid molecules (immunotoxins) for target cells. Immunol. Lett. 2, 97. 

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