Abrin P2 suppresses proliferation and induces apoptosis of colon cancer cells via mitochondrial membrane depolarization and caspase activation.

To explore the cytotoxic mechanism of abrin P2 on human colon cancer HCT-8 cells, abrin P2 was isolated from the seed of Abrus precatorius L. It was found that abrin P2 exhibited cytotoxicity toward 12 different human cancer cell lines. Our results demonstrated that abrin P2 suppressed the proliferation of human colon cancer cells (HCT-8 cells) and induced cell cycle arrest at the S and G2/M phases. The mechanism by which abrin P2 inhibited cell proliferation was via the down-regulation of cyclin B1, proliferating cell nuclear antigen and Ki67, as well as the up-regulation of P21. In addition, abrin P2 induced a dose- and time-dependent increase in the rate of HCT-8 cell apoptosis. Treatment with both Z-VAD-FMK, a broad-spectrum caspase inhibitor, and abrin P2 demonstrated that abrin P2 induced HCT-8 cell apoptosis via the activation of caspases. Together, our results revealed that abrin P2-induced apoptosis in HCT-8 cells was associated with the activation of caspases-3/-8/-9, the reduction in the Bcl-2/Bax ratio, the loss of mitochondrial membrane potential, and the increase in cytochrome c release. We further showed that abrin P2 administration effectively suppressed the growth of colon cancer xenografts in nude mice. This is the first report that abrin P2 effectively inhibits colon cancer cell growth in vivo and in vitro by suppressing proliferation and inducing apoptosis.

Inhibition of protein synthesis leading to unfolded protein response is the major event in abrin-mediated apoptosis.

Abrin obtained from the plant Abrus precatorius inhibits protein synthesis and also triggers apoptosis in cells. Previous studies from our laboratory suggested a link between these two events. Using an active site mutant of abrin A-chain which exhibits 225-fold lower protein synthesis inhibitory activity than the wild-type abrin A-chain, we demonstrate in this study that inhibition of protein synthesis induced by abrin is the major factor triggering unfolded protein response leading to apoptosis. Since abrin A-chain requires the B-chain for internalization into cells, the wild-type and mutant recombinant abrin A-chains were conjugated to native ricin B-chain to generate hybrid toxins, and the toxic effects of the two conjugates were compared. The rate of inhibition of protein synthesis mediated by the mutant ricin B-rABRA (R167L) conjugate was slower than that of the wild-type ricin B-rABRA conjugate as expected. The mutant conjugate activated p38MAPK and caspase-3 similar to its wild-type counterpart although at later time points. Overall, these results confirm that inhibition of protein synthesis is the major event contributing to abrin-mediated apoptosis.

The Fas/Fas ligand apoptotic pathway is involved in abrin-induced apoptosis.

Abrin is a plant glycoprotein toxin from the seeds of Abrus precatorius, sharing similarity in structure and properties with ricin. Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 µg/kg, causing death after accidental or intentional poisoning. It is a potent biological toxin warfare agent. There is no chemical antidote available against the abrin. The elucidation of molecular mechanism of abrin-induced cell death is important for development of therapy. Intrinsic pathway-mediated apoptosis has been well established in abrin-induced cell death. However, the detailed mechanism especially extrinsic receptor-mediated pathway remains uncharacterized. To assess whether some of the apoptosis known to occur after abrin exposure might be mediated by Fas/Fas ligand (Fas L) interactions, we analyzed effect of abrin on Fas pathway in Jurkat cells. Here, we report that activation of the Fas pathway is involved in abrin-induced apoptosis. Following treatment of abrin, Fas L was induced, which stimulated the Fas pathway by cross-linking Fas receptor (Fas R). Apoptosis was mediated by cleavage of the Fas R proximal caspase-8 and the downstream caspase-3, resulting in activation of the prototype caspase substrate poly-(ADP-ribose) polymerase and caspase-activated DNase. Blocking Fas L/Fas R interaction by using Fas inhibitor reduced abrin-induced apoptosis, further confirms involvement of Fas pathway. Activation of components of Fas pathway and caspases upon abrin treatment was also found in splenocytes in mice. Our findings offer new perspective for understanding the fundamental mechanism in abrin-induced apoptotic mechanism and may have implication in developing novel therapeutic strategies in the management for abrin-induced complications.

Abrin immunotoxin: targeted cytotoxicity and intracellular trafficking pathway.

BACKGROUND: Immunotherapy is fast emerging as one of the leading modes of treatment of cancer, in combination with chemotherapy and radiation. Use of immunotoxins, proteins bearing a cell-surface receptor-specific antibody conjugated to a toxin, enhances the efficacy of cancer treatment. The toxin Abrin, isolated from the Abrus precatorius plant, is a type II ribosome inactivating protein, has a catalytic efficiency higher than any other toxin belonging to this class of proteins but has not been exploited much for use in targeted therapy. METHODS: Protein synthesis assay using (3)[H] L-leucine incorporation; construction and purification of immunotoxin; study of cell death using flow cytometry; confocal scanning microscopy and sub-cellular fractionation with immunoblot analysis of localization of proteins. RESULTS: We used the recombinant A chain of abrin to conjugate to antibodies raised against the human gonadotropin releasing hormone receptor. The conjugate inhibited protein synthesis and also induced cell death specifically in cells expressing the receptor. The conjugate exhibited differences in the kinetics of inhibition of protein synthesis, in comparison to abrin, and this was attributed to differences in internalization and trafficking of the conjugate within the cells. Moreover, observations of sequestration of the A chain into the nucleus of cells treated with abrin but not in cells treated with the conjugate reveal a novel pathway for the movement of the conjugate in the cells. CONCLUSIONS: This is one of the first reports on nuclear localization of abrin, a type II RIP. The immunotoxin mAb F1G4-rABRa-A, generated in our laboratory, inhibits protein synthesis specifically on cells expressing the gonadotropin releasing hormone receptor and the pathway of internalization of the protein is distinct from that seen for abrin.

Reductive activation of type 2 ribosome-inactivating proteins is promoted by transmembrane thioredoxin-related protein.

Members of the type 2 ribosome-inactivating proteins (RIPs) family (e.g. ricin, abrin) are potent cytotoxins showing a strong lethal activity toward eukaryotic cells. Type 2 RIPs contain two polypeptide chains (usually named A, for "activity", and B, for "binding") linked by a disulfide bond. The intoxication of the cell is a consequence of a reductive process in which the toxic domain is cleaved from the binding domain by oxidoreductases located in the lumen of the endoplasmic reticulum (ER). The best known example of type 2 RIPs is ricin. Protein disulfide isomerase (PDI) was demonstrated to be involved in the process of ricin reduction; however, when PDI is depleted from cell fraction preparations ricin reduction can still take place, indicating that also other oxidoreductases might be implicated in this process. We have investigated the role of TMX, a transmembrane thioredoxin-related protein member of the PDI family, in the cell intoxication operated by type 2 RIPs ricin and abrin. Overexpressing TMX in A549 cells resulted in a dramatic increase of ricin or abrin cytotoxicity compared with control mock-treated cells. Conversely, no difference in cytotoxicity was observed after treatment of A549 cells or control cells with saporin or Pseudomonas exotoxin A whose intracellular mechanism of activation is not dependent upon reduction (saporin) or only partially dependent upon it (Pseudomonas exotoxin A). Moreover, the silencing of TMX in the prostatic cell line DU145 reduced the sensitivity of the cells to ricin intoxication further confirming a role for this enzyme in intracellular ricin activation.

Signaling different pathways of cell death: Abrin induced programmed necrosis in U266B1 cells.

Abrin is a type II ribosome-inactivating protein comprising of two subunits, A and B. Of the two, the A-subunit harbours the RNA-N-glycosidase activity and the B subunit is a galactose specific lectin that enables the entry of the protein inside the cell. Abrin inhibits protein synthesis and has been reported to induce apoptosis in several cell types. Based on these observations abrin is considered to have potential for the construction of immunotoxin in cell targeted therapy. Preliminary data from our laboratory however showed that although abrin inhibited the protein synthesis in all cell types, the mode of cell death varied. The aim of the present study was therefore to understand different death pathways induced by abrin in different cells. We used the human B cell line, U266B1 and compared it with the earlier studied T cell line Jurkat, for abrin-mediated inhibition of protein translation as well as cell death. While abrin triggered programmed apoptosis in Jurkat cells in a caspase-dependent manner, it induced programmed necrosis in U266B1 cells in a caspase-independent manner, even when there was reactive oxygen species production and loss of mitochondrial membrane potential. The data revealed that abrin-mediated necrosis involves lysosomal membrane permeabilization and release of cathepsins from the lysosomes. Importantly, the choice of abrin-mediated death pathway in the cells appears to depend on which of the two events occurs first: lysosomal membrane permeabilization or loss of mitochondrial membrane potential that decides cell death by necrosis or apoptosis.

Chimeric protein ABRaA-VEGF121 is cytotoxic towards VEGFR-2-expressing PAE cells and inhibits B16-F10 melanoma growth.

It has been known that VEGF(121) isoform can serve as a carrier of therapeutic agents targeting tumor endothelial cells. We designed and constructed synthetic cDNA that encodes a chimeric protein comprising abrin-a (ABRaA) toxin A-chain and human VEGF(121). Expression of the ABRaA-VEGF(121) chimeric protein was carried out in E. coli strain BL21(DE3). ABRaA-VEGF(121) preparations were isolated from inclusion bodies, solubilized and purified by affinity and ion-exchanged chromatography (Ni-agarose and Q-Sepharose). Finaly, bacterial endotoxin was removed from the recombinant protein. Under non-reducing conditions, the recombinant protein migrates in polyacrylamide gel as two bands (about 84 kDa homodimer and about 42 kDa monomer). ABRaA-VEGF(121) is strongly cytotoxic towards PAE cells expressing VEGFR-2, as opposed to VEGFR-1 expressing or parental PAE cells. The latter are about 400 times less sensitive to the action of this fusion protein. The biological activity of the ABRaA domain forming part of the chimeric protein was assessed in vitro: ABRaA-VEGF(121) inhibited protein biosynthesis in a cell-free translation system. Preincubation of ABRaA-VEGF(121) with antibody neutralizing the biological activity of human VEGF abolished the cytotoxic effect of the chimeric protein in PAE/KDR cells. Experiments in vivo demonstrated that ABRaA-VEGF(121) inhibits growth of B16-F10 murine melanoma tumors.

[Ribosome-inactivating lectins from plants].

There is a heterogeneous group of plant proteins which are able to enzymatically inactivate ribosomes by depurination of an invariant adenine from the 28 S ribosomal RNA. Some of these proteins are heterodimers having a lectin subunit which is joined by disulfide bond to the enzymatic subunit. Ricin and abrin which are among the most toxic substances known belong to the last group. This review focuses on the structure of the heterodimeric plant ribosome-inactivating proteins, the way of their action on ribosome, biosynthesis, intracellular trafficking, and their possible usage in medicine.

Effect of abrin on cell-mediated immune responses in mice.

Effect of abrin isolated from Abrus precatorius on the cellular immune responses was studied in normal as well as tumor-bearing animals. Administration of abrin was found to enhance the proliferation of splenocytes and thymocytes (lymphocytes in general) in responses to mitogens. Natural killer cell activity was enhanced significantly by abrin in both the normal (49.8% cell lysis on day 9) and the tumor-bearing group (51.7% cell lysis on day 9), and it was found to be earlier than the control. Antibody dependent cellular cytotoxicity was enhanced in the abrin treated tumor-bearing group on the ninth day (44% cell lysis). An early antibody dependent complement mediated cytotoxicity was observed in the abrin treated group on day 15 (27.6% cell lysis). Results of our present study suggest the immunomodulatory property of abrin.

Abrin induces HeLa cell apoptosis by cytochrome c release and caspase activation.

We identified apoptosis as being a significant mechanism of toxicity following the exposure of HeLa cell cultures to abrin holotoxin, which is in addition to its inhibition of protein biosynthesis by N-glycosidase activity. The treatment of HeLa cell cultures with abrin resulted in apoptotic cell death, as characterized by morphological and biochemical changes, i.e., cell shrinkage, internucleosomal DNA fragmentation, the occurrence of hypodiploid DNA, chromatin condensation, nuclear breakdown, DNA single strand breaks by TUNEL assay, and phosphatidylserine (PS) externalization. This apoptotic cell death was accompanied by caspase-9 and caspase-3 activation, as indicated by the cleavage of caspase substrates, which was preceded by mitochondrial cytochrome c release. The broad-spectrum caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVADfmk), prevented abrin-triggered caspase activation and partially abolished apoptotic cell death, but did not affect mitochondrial cytochrome c release. These results suggest that the release of mitochondrial cytochrome c, and the sequential caspase-9 and caspase-3 activations are important events in the signal transduction pathway of abrin-induced apoptotic cell death in the HeLa cell line.

Protein disulphide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum.

Cells expressing ricin B chain within the secretory pathway are significantly more resistant to intoxication by ricin holotoxin but not to other cytotoxins that exploit similar endocytic routes to the cytosol. Furthermore, cells expressing the related B chain of abrin are protected against both incoming abrin and ricin. These phenotypes can be correlated with the abilities of the respective B chains to form disulphide-linked A-B holotoxins, since abrin B chain forms heterodimers with either abrin or ricin A chains, whereas ricin B chain forms heterodimers with ricin A chain only. In the ricin B-expressing cells, this newly made lectin disappears with biphasic kinetics comprising a retention phase followed by slow turnover and disposal after disengagement from calnexin cycle components. Interference with ricin cytotoxicity occurs during the early retention phase when ricin B chain is associated with PDI (protein disulphide-isomerase). The data show that retrotranslocation of incoming toxin is impeded by PDI-catalysed formation of heterodimers between endogenous B and A chains derived from reduced holotoxin, thus proving that reduction of ricin occurs in the endoplasmic reticulum. In contrast with other toxins, ricin does not appear to require either proteolytic cleavage or unfolding for PDI-catalysed reduction.

Plant-derived abrin-a induces apoptosis in cultured leukemic cell lines by different mechanisms.

Abrin-a consists of A-chain with N-glycosidase activity, which inhibits protein synthesis, and lectin-like B-chain responsible for binding with cell-surface receptors and penetrating of abrin-a molecule into the cells. As a lectin component, the B-chain can also participate in cell signal transduction. It has been reported that abrin induces apoptosis, but the molecular mechanism(s) of this induction have been obscure and several alternative variants have been discussed. The present study demonstrates that abrin-a induces apoptosis in human cultured cell lines, derived from acute lymphoblastic leukemia (ALL) (Jurkat, CCRF-CEM, MOLT-4, HPB-ALL). The apoptosis was estimated by: phosphatidylserine (PSer) exposure at the cell surface, activation of caspase cascade, and DNA fragmentation. The penetrating of abrin-a into the cells was detected by fluorescent confocal microscopy, using fluorescein isothiocyanate (FITC) as a fluorescent marker. It was established that the effect of abrin-a on the apoptosis induction in leukemic cells was dose- and time-dependent. The process was initiated 1 h after abrin-a application (before its penetrating into the cells) and was characterized with PSer translocation from the inner to the outer monolayer of plasma membrane, caspase activation on the first to second hour after beginning of treatment, with maximum on the third to fourth hour, and DNA fragmentation on the fourth to sixth hour, depending of the cell line. The exposure of PSer on the cell surface was detected in Jurkat, CCRF-CEM, and MOLT-4 cells. In HPB-ALL, no significant changes in PSer exposure on the cell surface was observed. Activation of caspase-3, -8, and -9 was detected in Jurkat, MOLT-4, and HPB-ALL. Surprisingly, the activity of caspase-3 increased on the first hour after beginning of treatment, while the activity of caspase-8 and -9 began to increase on the second hour. In CCRF-CEM, activation of caspases was not measured, but the apoptosis progressed to DNA fragmentation in a dose- and time-dependent manner. DNA fragmentation was also detected in Jurkat, but not in MOLT-4 and HPB-ALL cells. It seems that the mechanisms of abrin-a-induced apoptosis are different and the progress of apoptosis depends of the cell line. There was a very good positive correlation between the agglutinating activity of abrin-a and development of apoptosis to DNA fragmentation. The time-dependent effects of abrin-a on apoptosis as well as its time-dependent penetration into the cells suggest that the B-chain probably triggers the apoptosis, while the A-chain and breakage of the disulfide bond are responsible for its progress.

Cloning, expression of the abrin-a A-chain in Escherichia coli and measurement of the biological activities in vitro.

The coding sequence of abrin-a A-chain (ABRaA) gene was obtained by RT-PCR and cloned into the expression vector pET28b. The mature ABRaA has been highly expressed in the cytoplasm of Escherichia coli by 1 mmol/L IPTG induction, and the yield of the soluble recombinant protein was 4 mg/L of induced culture. The recombinant ABRaA was purified to be homogeneity. The biological activities of expressed ABRaA were demonstrated in vitro. It strongly inhibited the protein biosynthesis of rabbit reticulocyte lysates, with an IC(50) of 0.08 nmol/L. It also depurinated 28 S rRNA through cleaving at the A4324 site in rat liver ribosomes by its N-glycosidase activity. These data suggested that the recombinant ABRaA could be used for the preparation of immunotoxins as a potential cancer chemotherapeutic agent.

Antitumour effect of abrin on transplanted tumours in mice.

Abrin, a galactose specific lectin was purified using sepharose 4B affinity column from seeds of Abrus precatorius. It exhibited antitumour activity in mice when used at a sublethal dose of 7.5 micrograms/kg every alternate day for 10 days. Both intralesional and intraperiloneal (i.p.) administration of abrin was effective in reducing solid tumour mass development induced by Dalton's Lymphoma Ascites (DLA) and Ehrlich's Ascites Carcinoma (EAC) cells. DLA cell line was more sensitive to abrin than EAC. Abrin when injected i.p. increased the life span of ascites tumour bearing mice. Abrin when used simultaneously with tumour cells brought about maximum antitumour effect. On developed tumour masses, abrin administration brought about significant reduction in tumour volume, especially in DLA induced tumours. Prophylactic administration of abrin was found ineffective.

Immunopotentiating activity of abrin, a lectin from Abrus precatorius Linn.

A non-toxic dose of abrin, (1.25 microg/kg body wt) consecutively for five days in normal mice stimulated specific humoral responses. A noticeable increase was observed in total leucocyte count, lymphocytosis, weights of spleen and thymus, circulating antibody titre, antibody forming cells, bone marrow cellularity and alpha-esterase positive bone marrow cells. The results suggest that abrin can potentiate the humoral immune response of the host.

Abrin triggers cell death by inactivating a thiol-specific antioxidant protein.

Abrin A-chain (ABRA) inhibits protein synthesis by its N-glycosidase activity as well as induces apoptosis, but the molecular mechanism of ABRA-induced cell death has been obscure. Using an ABRA mutant that lacks N-glycosidase activity as bait in a yeast two-hybrid system, a 30-kDa antioxidant protein-1 (AOP-1) was found to be an ABRA(E164Q)-interacting protein. The interaction was further confirmed in vitro by a glutathione S-transferase pull-down assay. The colocalization of endogenous AOP-1 and exogenous ABR proteins in the cell was demonstrated by confocal immunofluorescence. We also demonstrated that ABRA attenuates AOP-1 antioxidant activity in a dose-dependent manner and the intracellular level of reactive oxygen species (ROS) increases in ABR-treated cells. Moreover, ROS scavengers N-acetylcysteine and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl delayed programmed cell death. This indicates that ROS are important mediators of ABR-induced apoptosis. When ectopically expressed, AOP-1 blocked the release of cytochrome c and prevented apoptosis in ABR-treated cells. These findings suggest that the binding of ABRA to AOP-1 promotes apoptosis by inhibiting the mitochondrial antioxidant protein AOP-1, resulting in the increase of intracellular ROS and the release of cytochrome c from the mitochondria to the cytosol, which activates caspase-9 and caspase-3.

Biological activities of the lectin, abrin-a, against human lymphocytes and cultured leukemic cell lines.

The cytoagglutination by abrin-a against human cultured cell lines derived from acute lymphoblastic leukemia (ALL) and human peripheral blood lymphocytes obtained from normal adults and from patients with adult T cell leukemia (ATL) was investigated. Among acute T lymphoblastic leukemia (T-ALL) cell lines, abrin-a showed strong cytoagglutination against relatively differentiated cell lines, such as Jurkat and CCRF-HSB-2. Among acute B lymphoblastic leukemia (B-ALL) cell lines, abrin-a strongly agglutinated an immature cell line, NALM6. In comparison with ALL cell lines, cytoagglutination by abrin-a against normal lymphocytes was weak. Abrin-a showed higher cytoagglutination against lymphocytes derived from ATL than lymphocytes derived from normal adults. In connection with the cytoagglutination, abrin-a-induced cytotoxicity against human cultured leukemic cell lines was evaluated. In proportion to the extent of cytoagglutination, abrin-a induced cytotoxicity in Jurkat, CCRF-HSB-2, MOLT-4, RPMI8402, and BALL-1 as well. Although CCRF-CEM and BALM-1 were both weakly agglutinated by abrin-a, these cell lines were very sensitive to the abrin-a-induced cytotoxicity. NALM6 was strongly agglutinated by abrin-a, but abrin-a exhibited less strong cytotoxicity against this cell line. These results suggest the feasible application of abrin-a as a tool to distinguish the human leukemic cells and its potential for clinical application.

Effect of molluscicidal components of Abrus precatorius, Argemone mexicana and Nerium indicum on certain biochemical parameters of Lymnaea acuminata.

Exposure to 40% and 80% of the 24 h LC50 of the molluscicidal component of Abrus precatorius (abrin and glycyrrhizin), Argemone mexicana (protopine and sanguinarine) and Nerium indicum (oleandrin) caused a significant decrease in the levels of protein, free amino acid, DNA and RNA in the nervous tissue of Lymnaea acuminata. Except for glycyrrhizin, all the above molluscicides caused a significant reduction in phospholipid levels and a simultaneous increase in the rate of lipid peroxidation in the nervous tissue of treated snails.