Ribosome-inactivating Proteins (Ricin and Related Proteins) || A Long Journey to the Cytosol

Ribosome-inactivating Proteins: Ricin and Related Proteins, First Edition. Edited by Fiorenzo Stirpe and Douglas A. Lappi.

© 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.

161

11 A Long Journey to the Cytosol: What do We Know about Entry of Type 1 RIPs Inside a Mammalian Cell?Rodolfo Ippoliti1 and Maria Serena Fabbrini2

1 Department of Life, Health and Environmental Sciences, University of L’Aquila, Italy2 Ministry of Instruction, University and Research (MIUR), Italy

Introduction

Ribosome-inactivating Proteins (RIPs) From Plants: What Are They?

Many plant species produce ribosome-inactivating proteins (RIPs)1 that are classified into two main groups, depending on their structure. Type 1 RIPs are monomeric proteins,2 constituted by a 30-kDa polypeptide, usually with a high content of basic amino acids conferring to the toxin a high IP (>9). Type 2 RIPs are instead made of two different chains, linked together by a disulfide bond, one being the catalytic toxic A chain, while the other, the (B)inding chain, is a lectin allowing the holotoxin to bind exposed cell surface glycoproteins or glycolipids in mammalian cell membranes. Type 2 RIPs are neutral proteins that are very poor in lysines, thus greatly differing from type 1 RIPs. They both act by depurination of a single adenine (A4324 in rat), thus irreversibly blocking protein synthesis3, 4 showing an N-glycosidase activity (see also Chapter 10). Recently, substrates other than rRNA were reported to be depurinated, such as DNA, mRNA, or viral RNAs by type 1 RIPs.5–7 Most of the latter activities were measured in vitro on isolated substrates with higher efficiencies obtained at acidic pH values, thus the significance of these data in vivo remains to be firmly established. Several type 1 RIPs were reported to remove more than one adenine residue per ribosome, possessing a polynu-cleotide : adenosine glycosidase (PNAG) activity. However, the activity of seed-extracted saporin (from Saponaria officinalis) was also found to vary between different experiments and batches when poly(A) or viral RNAs were used,8 while recombinant single saporin isoforms9 were instead found to be essentially inactive on the latter but active on rRNA at pH 4.0. Saporin-6 released the highest number of adenine molecules from rat ribosomes and poly(A), efficiency on herring sperm DNA was found to be similar to that of dianthin 30, bouganin, and PAP-R, while saporin-6 isoform was found to be the most active toxin in inhibiting protein synthesis. An independent study reported recently that seed-extracted saporin could release a single adenine from 80S ribosomes,10 confirmed by rRNA found to be depurinated at a single site, most likely corresponding to the alpha sarcin-ricin target,11 when rRNA was extracted from intoxicated mammalian cells exposed to this same toxin.

162 RIBOSOME-INACTIVATING PROTEINS

A commonly accepted view is that these proteins may confer an advantage to the RIP-expressing plants7 having potentially antifungal, antibacterial, or broad-spectrum anti-viral activities, although the exact mechanism(s) as to how this can be achieved are to be fully elucidated. We will not report on type 3 plant RIPs produced as inactive pre-pro enzymes, activated by proteolytic cleavage of specific residues within the catalytic site,12 as the number of members is quite limited and not yet used for targeting purposes.

Usually, native RIP proteins can be purified as mature polypeptides in planta as for the most popular type 1 RIPs: saporin, momordin, gelonin, PAP, trichosanthin, and PDL.13–20

Recently, type 1 plant RIPs have been investigated as new potential anticancer agents, especially now that many cDNAs are available for designing recombinant fusion constructs21–46 or for gene therapy approaches.47 A next step forward in these targeting approaches implies the need to modify the RIP coding genes for expressing targeted chimeric constructs recombinantly. From this perspec-tive, knowledge about the structure of the desired RIP and its properties of absorption by mamma-lian cells is fundamental for the production of recombinant toxins that may efficiently kill target cancer cells both in vitro and in vivo.

Tridimensional Structures and Catalytic Active Residues

To better characterize the intracellular route of type 1 RIPs, we must briefly mention what is known about the structure of these toxins (for further details on the structure of these classes of enzymes, please refer to Chapter 8).

The catalytic chains of the two main classes of RIPs share little homology in terms of amino acidic sequence, although key residues at the catalytic cleft are well conserved and their three- dimensional fold can be superimposed. In Figure 11.1 we report the alignment of six among the most studied type 1 RIPs.

Despite differences in amino acid sequences, the three-dimensional structures of all these pro-teins are superimposable48–53 with the catalytic residues being highly conserved among all toxins belonging to the RIP family (Figure 11.2) and maintaining the typical N-glycosidase fold that is found conserved also among similar bacterial enzymes.

As will be described below, the intracellular pathways of RIPs may significantly differ, as indi-cated by a broad range of effects seen with chemical enhancers, suggesting that amino acid differ-ences rather than specific structural motifs may underly the differences in behavior.

Direct Binding of Type 1 RIPs to Mammalian Cell Membranes

Type 1 RIPs are supposed to enter cells only via fluid-phase endocytosis or macro-pynocytosis.11, 54–56 Still, there is some evidence that specific binding to cell surface receptors may occur.

The abortifacient activity of saporin and other type 1 RIPs such as tricosanthin is well docu-mented,57–59 based on their extremely high toxicity towards human trophoblasts.60

Saporin may bind the LDL-related receptor protein (LRP)/alpha2-macroglobulin receptor (α2MG-R)61 in a variety of cell lines, including U937 monocyte-like cells. The receptor normally binds alpha 2-macroglobulin/proteinase complexes, plasminogen-activator-inhibitor complexes, apoE-enriched beta-very low density lipoproteins, and lipoprotein lipase. All these ligands are com-petitors of saporin binding since they decrease saporin toxicity.61 It has already been demonstrated that RTA covalently linked to α2MG can bind α2MG-R retaining its activity,62 thus suggesting a putative role for α2MG-R in mediating RIP internalization.

A LONG JOURNEY TO THE CYTOSOL 163

Figure 11.1 Amino acid sequence aligment of several type 1 plant RIPs. Catalytic residues are evidenced in boxes.

Figure 11.2 3D-structures of type 1 RIPs superimposed to Ricin A chain (RTA). (a) Color codes: RTA grey; Saporin green;

Dianthin cyan; Gelonin magenta; Luffin yellow; Momordin light orange; Pap blue. (b) Residues shown inside the catalytic site are

numbered following Saporin sequence. (This figure also appears in the color plate section.)

(a)

W206

(b)

Y72

R179

E176 Y120


Furthermore, α2MG-R/LRP is the receptor mediating Pseudomonas aeruginosa exotoxin A binding and internalization,63 as well as being a fundamental receptor to elicit immunological responses due to its opsonization activity that can lead foreign proteins to be adsorbed by antigen-presenting cells or macrophages.64–66 Indeed, both saporin and RTA have been reported to be very potent immunogens67–69 indicating their interaction with immune cells.

So far, α2MG-R is the only receptor identified as binding directly to single-chain type 1 RIPs.70, 71 α2MG-R may also play a crucial role in the cellular absorption mechanism of a toxic chimera. Indeed, binding and internalization of urokinase receptor-targeted saporin (uPA-sap) or recombinant saporin fused to human uPA amino-terminal fragment (ATF-saporin) was demonstrated to be also dependent on the presence of saporin, interacting with the α2MG-R.71 Physiologically, α2MG-R can be internalized via a quartenary complex with uPAR-bound uPA complexed with its inhibitors, that is PAI1,72 since the ATF domain is unable to internalize. It was therefore suggested that saporin could mimic PAI1 moiety in triggering the internalization of ATF–saporin or uPA–saporin conju-gates. Partially supporting this view, we have reported that the uPA–saporin conjugate is indeed internalized in mouse fibroblasts over-expressing the human uPA receptor (uPAR), but it follows a peculiar two-step endocytic mechanism: the chimera is initially concentrated at the cell surface by interaction with uPAR but internalizes via α2MG-R alone.73

Bagga et al.74 reported controversial experiments describing a non-complete dependence on α2MG-R for saporin internalization in a variety of cell lines, suggesting that this toxin does not exclusively use this receptor to gain access to the cell. It must be underlined, however, that the α2MG/ LDL receptor family includes seven closely related family members: LDLR, the very-low-density lipoprotein (VLDL) receptor, apoE receptor 2, multiple epidermal growth factor-like domains 7 (MEGF7), glycoprotein 330 (gp330/megalin/LRP2), LRP1, and LRP1B, that have been shown to be promiscuous in ligand binding.75 For instance, when mouse embryonic fibroblasts (MEF-2) derived from LRP1 knock-out mice were exposed to saporin, they showed 10-fold less sensitivity to the toxin, as compared to MEF1 cells carrying both α2MG-R/ LRP1 and low-density lipoprotein receptor (LDLR); this possibly indicates a role for LDLR, or other family members as well, in mediating saporin internalization, at least in mouse fibroblasts.11

Finally, there is strong evidence that type 1 RIP trichosanthin (TC) can interact with cells via LRP and megalin receptors.76 TC is active on trophoblasts7 which express LRP and on tubular epithelial cells which express megalin receptors. Thus, LRP family members appear to be a diffused system putatively able to mediate RIP’s internalization, although this has not yet been investigated for all type 1 toxins.

Possible Intracellular Sorting/Delivery Routes Established for RIPs by Biochemical Methods

Saporin and trichosanthin, as ricin holotoxin, may thus enter cells by endocytosis-mediated inter-nalization, but these toxins must then escape from the endomembrane system to reach their targets, that is the cytosolic ribosomes. The latter process represents the rate-limiting step and thus may directly affect the toxin potency.

In the case of ricin, a number of independent studies have essentially clarified the major steps that the dimeric toxin undergoes while traveling backwards along the secretory route: from the trans-Golgi network to the endoplasmic reticulum (ER) where the SS bond linking A to B chain is dis-rupted by PDI77 and possibly one (or only a few) molecule(s) of RTA is disguised as a misfolded protein to exploit ER-associated degradation (ERAD) pathways and be retro-translocated into the cytosol where the proteasomes are (major details on the entire process and original studies can be


found in this book in Chapter 6). Indeed, appending an ER-retrieval signal such as KDEL (amino acid one letter code) to ricin or RTA may facilitate this retrograde transport route and thus sensitize the cells 10- to several 100-fold towards the KDEL-bearing toxin.55, 78

Type 1 RIP Internalization via Endosomal/Lysosomal System?

A major step forward in assessing the main differences between ricin and saporin intracellular routes was presented in a study where, in Vero cells, we documented that two known treatments, Brefeldin A protecting the cells against ricin intoxication (by disrupting Golgi stacks) and chloroquine, a lyso-somotropic agent enhancing endosomal pH and thus, able to protect cells against diphtheria toxin (DT), had no effects on saporin.11 DT that directly escapes from acidified endosomes to the cytosol, relying upon a peculiar acid-induced pore formation conformational step to facilitate its own export,79–85 was used as a control. Furthermore, when a KDEL signal was appended to either saporin or recombinant ATF-saporin generating ATF-saporin KDEL it had little effect or even showed a decreased cytotoxicity of the latter, as compared to the parental toxins.11 This was, indeed, fully con-sistent with our previous findings: while tracing a ProuPA-saporin chimera fluorescently labeled with TRIC, it was nicely excluded from Golgi stacks after internalization but found mostly in punctuate structures, overlapping with endosomes.73 Saporin, in contrast to ricin, also could not be localized to the Golgi after internalization, but could co-localize in early endosomes with the EEA1 marker.11

In collaboration with Teter’s group, we more recently showed that cell exposure to dimethylsulfox-ide (DMSO) after saporin entry or lipopolyamine co-administration may generate a substantial cel-lular sensitization towards saporin or uPA-saporin.86 In addition, by using lysosome-endosome CHO mutant cell lines (LEX1 and LEX2), we could demonstrate that in the case of saporin (but not RTA) an endosomal trafficking step preceding cargo delivery to a late endosomal compartment was indeed taking place. This was presumably the major site for the DMSO-enhanced entry of saporin into the cytosol. No toxin sensitization could be observed for DMSO or lipopolyamine-treated cells exposed either to diphtheria toxin or to ricin holotoxin, indicating that this effect was peculiar to the saporin entry route, implying a clear role for early and late endosomal membranes in mediating this step.

More recently, another natural (triterpenoid) compound, saponin, has been shown to have intriguing effects on saporin-based chimeras; and the group of Fuchs and Bachran described the selective potentiation induced by saponins87 from Saponinum album or Gypsophila paniculata whose activity is hypothesized to act specifically on type 1 RIPs such as saporin by inducing their facilitated insertion within endomembranes.88 Lipid partitioning studies using Triton X-114 demon-strated that while RTA is predominantly found in the detergent phase, the ricin B chain with the holotoxin and several type 1 RIPs, including saporin, are instead found in the aqueous phase.89 RTA may interact with negatively-charged lipid vesicles and with ER membranes, undergoing a confor-mational change that could make it a better substrate for the ERAD system.90

Some data further support the view that certain type 1 RIPs are able to interact with endosomal membranes by inducing a transient modification allowing direct release of the toxin into the cell cytoplasm. This behavior has been also suggested by in vitro experiments studying the effect of RIPs on the stability of artificial vesicles (liposomes) containing negatively-charged lipids. Trichosanthin91, 92 was demonstrated to induce fusion and leakage of negatively-charged liposomes, this phenomenon being attributed to its C-terminal region.93 Similarly, saporin could act on liposomes94 and also, in this case, its C-terminal region was identified as fundamental for the in vivo enzymatic activity.95 Thus, at least for the latter toxins, their release from endocytic vesicles may be mediated in part by binding to negatively-charged membranes, thanks to both electrostatic and hydrophobic interactions.


In the case of RTA, it has been demonstrated it undergoes major structural rearrangement upon interaction with liposomes89 as cinnamomin catalytic A chain,96 an indication that A chains from type 2 RIPs may have greater flexibility, allowing them to use different mechanisms of translocation.

RIP Internalization via Golgi/ER

The lectin B chain domain allows type 2 toxins to efficiently bind to eukaryotic cells, triggering receptor-mediated endocytosis and mediating their retrograde transport (i.e., ricin, abrin, and modec-cin) to the (ER) where the SS bond linking the B to the A chains undergoes reduction just before the A chain is released into the cytosol,77, 97, 98 despite the report that a ricin variant containing A and B chains linked by a non-reducible covalent bond could still be as active against eukaryotic cells. 99

Why don’t type 1 RIPs follow this same route? Their isoelectric points are extremely high, for instance the pI calculated for SAP is almost 10. Saporin accounts for almost 10% of lysine residues, while RTA has only two lysines and a few arginine residues. Thus, the overall biochemical differ-ences between the two RIP classes likely also reflect their different cytosolic entry routes. Indeed, RTA, as most of the catalytic A chains of type 2 RIPs, shows isoelectric points predominantly in the acidic or neutral pH ranges. This would seem important since, for example, proteins found in the ER tend to have mostly acidic pI values.100, 101 It was earlier suggested that the A chains of type 2 RIPs have been under selective pressure to lower lysine content while proteins that do not use the retrograde transport route, such as DT, display a high lysine residue content.102, 103 Lysine is one of the possible targets of ubiquitynilation, a signal targeting ERAD substrates to proteasomes for destruction. It has indeed been demonstrated that by reducing the lysine content, RTA increases in stability in the cell cytoplasm103 because it avoids proteasome-mediated degradation while, in con-trast, RTA with extra lysines displayed much less toxicity than the wild-type holotoxin.103 A fusion of type 1 momordin (pI = 9.3) to the ricin B chain resulted in a chimeric toxin that undergoes an inefficient intracellular entry, showing a greatly decreased potency as compared to native ricin.104

Concerning the target, negative electrostatic potential has been reported to cover most of the ribosomal-exposed surfaces, possibly arising from the RNA negatively-charged phosphodiester backbone and conserved solvent-exposed acidic patches of ribosomal proteins, suggesting an evo-lutionary conservation among the ribosome’s overall electrostatics. Interestingly, ricin A, saporin, and gypsophilin have been shown to depurinate ribosomes with a strong salt dependence, implying that RIPs may all share a common mechanism of electrostatically-facilitated ribosomal targeting.105 Despite these findings, as a further independent confirmation of ricin productive passage through the ER, some new compounds have been identified which could protect against ricin poisoning. They are called retro 1 and retro 2 and are the result of screening a total of 160,000 chemical com-pounds for blocking the retrograde transport route to the cytosol. They have been demonstrated to inhibit the passage of the ricin molecules from late endosomes to TGN, without affecting the retro-grade transport of other cellular cargo proteins. The effectiveness of these compounds could also prove very useful for Shiga toxin 1 fecal intoxications, since this bacterial RIP may follow, as ricin, a similar retrograde route to access cytosolic ribosomes.106

However, the Johannes group reported that Shiga toxin could not be found on the late endosomal membranes “en route” to the TGN,107 which led to the view that this bacterial toxin could directly traffic from early endosomes to the TGN. Clathrin, that also localizes onto early endosomes, was shown to be required for the retrograde sorting of Shiga toxin, because during inhibition of retro-grade tubule formation in clathrin-depleted cells, the toxin co-localized with transferrin receptors and could not reach the TGN/Golgi, thus clearly demonstrating its need for clathrin in order to leave


early endosomes.108 Very recently, it has also been suggested that Shiga toxin might also traffic from recycling endosomes to the TGN. Different itineraries for the retrograde route of Shiga toxin and TGN38 were demonstrated by use of a distinct retromer component, regulating the transport of these two model cargos.109 Retrograde trafficking routes from early, recycling, and late endosomes would presumably coexist in parallel, being possibly dynamically interconnected. Johannes and Wunder recently presented landmark contributions110 that would favor coexistence of these three retrograde sorting processes from early, recycling, and late endosomes concluding: “the situation is somewhat more complex and possibly more intricate than this.”

Exosome-Mediated Delivery

A novel intracellular transport mediated by exosomes was recently identified111 for trichosanthin (TC). Jurkatt T-cells (that do not express members of the LDL receptor family) were found to be essentially resistant to free TC; but became very sensitive to TC-loaded vesicles secreted by two target cell lines, JAR and K562, where part of the endocytosed toxin was found incorporated into “pomegrenade” vesicles, deriving from multivesicular body (MVB) membranes, and could be further secreted upon fusion of the MVB with the plasma membrane. Whether other type 1 RIPs could exploit this exosome-mediated trafficking route remains to be clarified. Since some gelonin chimeric fusions were found to be able to trigger mammalian cell death following an autophagy-mediated pathway,112–114 it would seem important to verify whether other type 1 RIPs are also able to hijack the autophagic or exosome-mediated pathways.

Figure 11.3 summarizes the three main recognized pathways in RIP internalization.

Identification of Endocytotic Compartments Containing RIPs During Intracellular Delivery

Despite the large number of papers describing the toxic properties of type 1 RIPs, only a few deal with the intracellular characterization of the toxin delivery. It has been reported115 that trichosanthin is accu-mulated in the endosomal/lysosomal system in choriocarcinoma cell lines, but may instead enter hep-atoma cells via membrane passive diffusion. In the first case, the toxin exploits LDL receptors to gain access to the cell cytoplasm. Among other internalization paths based on the direct interaction of glycosylated RIPs with the cellular membranes, two have been reported for gelonin116 and ricin A chain.117 Presence of mannose residues in these toxins allowed them to be adsorbed by macrophages via mannose receptors, triggering the endocytotic processes. RTA could be seen accumulated into late endosomes and lysosomes after binding, in macrophages, being partially degraded before reaching the cell cytoplasm. Furthermore, a fusion between RTA and GFP has been demonstrated to reach the Golgi complex after internalization, as a consequence of a passive fluid-phase absorption into cells.118

Saporin could be visualized inside endo/lysosomal compartments11 using a rhodamine-labeled toxin, but not in Golgi structures (as was RTA). Following internalization in Vero cells, a recombinant gelonin could also be localized inside endosomal and lysosomal vesicles; however, the addition of a polyarginine tail (R9) facilitated the release of the latter toxin from these compartments, behaving as a substrate for proteolytic activation.119 Saporin cytotoxicity varies dramatically between different mammalian cell lines, with concentrations inhibiting protein synthesis by 50% (IC

50) spanning at

least three orders of magnitude from the nanomolar to the micromolar range.70, 74 In the model cell line for toxin studies, Vero, IC

50 is about 10 nM, while in Hela cells it was as high as 2 μM.11 In fact,


Bolognesi and co-workers120 have seen that Saporin-S6 can be taken up by HeLa cells, presumably via fluid-phase pinocytosis, and made a characterization of saporin internalization followed by fluo-rescence and electron microscopy, reporting an accumulation of at least 30% of the toxin inside endosomes but only less than 10% visualized in the Golgi complex. Furthermore, in these condi-tions, they also reported a progressive accumulation of saporin inside perinuclear structures, an observation that the authors linked to the possible direct activity saporin may exert on nuclear DNA.

Binding of Targeted Type 1 RIPs to Cellular Membranes Through Selective Carrier Molecules

Since the difference in the internalization efficiency among type 1 RIPs, as compared to type 2 RIPs, is due to the absence of a selective B (binding) chain, several molecules have been linked or fused within recombinant molecules to enhance type 1 RIP toxicity towards specific target cells. Chimeric molecules are often obtained by linking a full antibody (often a monoclonal) or an antibody fragment

Extracellular space

Endosome

TGN

Golgi

ER

ERADRicin holotoxin

RTA

RTB

Saporin

Trichosanthin

Glycoprotein

LRP

Cytosol

Nucleus

MVB

A B

?

??

C

Exosomes

Figure 11.3 Intracellular pathways of RIPs (from de Virgilio M, Lombardi A, Caliandro R, Fabbrini MS. Ribosome-inactivating

proteins: from plant defense to tumor attack. Toxins. 2010;2: 2699–2737). (This figure also appears in the color plate section.)


(i.e., a scFv) to RIP domains, such as saporin, gelonin, or ricin A chain.121–126 Among targeting domains, those more widely used are growth factors such as transferrin,127, 128 epidermal growth factors,129, 130 and fibroblast growth factors,131 targeting receptors over-expressed in tumor cells.

We already reported in the section above our findings with the uPA and ATF–saporin chimeras, targeting urokinase receptors that mediate metastatic spread of several cancer cells. However, only a few examples of RIPs conjugated to carriers have been characterized for their intracellular traf-ficking. We have also analyzed routing of a transferrin–saporin conjugate as compared to ricin holotoxin or a transferrin–ricin A chain conjugate.128 By saporin and RTA fluorescent labeling, we could conclude that, as the B chain for ricin, transferrin leads the intracellular delivery of both RTA and saporin to endosomal compartments containing free transferrin (recycling/early endosomes).

Intriguingly, we also observed (Ippoliti et al., unpublished results) that saporin instead could be delivered to the Golgi complex when linked to a monoclonal antibody against the CD7 B marker, despite the fact that the latter immunotoxin did not show kinetics of internalization and cytotoxicities comparable to those of ricin, notwithstanding its significant co-localization within the Golgi stacks.

This may indicate that using different carrier molecules may lead to different alternative intracel-lular trafficking pathways, not always the ones best suited to allow the RIP moiety to efficiently reach the cytosolic compartment. This is a crucial issue that should be particularly taken into consideration when producing chimeric fusions to selectively target cancer cells.

Based on the characteristics of the A chains of type 2 or type 1 RIPs described above, one should consider that for construction of a chimeric protein. if the targeting carrier is thought to deliver the toxin domain to endo-lysosomal compartments, a type 1 RIP would be better suggested; in contrast a type 2 A chain should be the best choice for carriers targeting the TGN.

The Effect of Known Potentiating Agents in the Intracellular Delivery of Toxins and their Relationship with Intracellular Delivery Paths

Several chemical agents have been extensively studied as potential adjuvants of RIP cytotoxicity againts cancer cells and could influence type 1 RIP trafficking and toxicity. However, we must be aware that the concentrations used may interfere with more than one intracellular pathway for certain substances. Among these, alkalinizing agents of acidic compartments, such as chloroquine acting on lysosomes or the weak base NH4Cl (ammonium chloride), elicit an increase in cellular toxicity on a transferrin-targeted saporin,132, 133 whereas in contrast an antibody-targeted saporin was scarcely influenced134–136 or even showed a cellular-type dependence.137

In our hands, unconjugated saporin was not potentiated by chloroquine11 while ATF–saporin could show an increased toxicity following exposure of the target cells to chloroquine, possibly related to its protection against proteolytic degradation.71 Ricin was releasable at constant rates from endosomes isolated from intoxicated cells, independently from pH changes,138 whereas some ricin A-containing immunotoxins were found to be more active139 in the presence of chloroquine, ammonium chloride. or monensin.

Monensin is a carboxylic antibiotic ionophore exchanging sodium with protons, which may induce alkalinization of several intracellular compartments (including lysosomes or endosomes) and Golgi apparatus swelling, resulting in trans-Golgi network collapse. Therefore, monensin can modulate the toxicity of both toxins traveling along the Golgi or the endosomal system. Indeed, toxicity of the model toxins ricin140 and mistletoe lectin 1141 and that of a gelonin-containing immu-notoxin directed towards breast carcinoma cells142 were enhanced by this drug. Potentiation of saporin–transferrin conjugate activity by monensin was also obtained.132


The intriguing results reported above may suggest that for plant RIPs, the change in cytotoxicity observed with these agents may not only depend on the intracellular compartment reached by the chimeric toxin but may also be strongly determined by the carrier/targeting molecule. On the con-trary, chimeric toxins including diphtheria toxin all depend on passage through acidic vesicles, and in fact the latter, if treated with alkalinizing agents, shows a substantial reduction in toxicity.79, 143–145

While the precise mechanism of these substances remains fairly unclear, some may influence the stability of the chimeric toxins (those referred to as “lysosomotrophic” agents). Moreover, despite the fact that their effects in vitro were readily measurable, chloroquine and ammonium chloride failed to exert the same potentiating effect on in vivo treatments in combination with immunotoxins.146 For instance, since phosphoinositide 3-kinase (Pl 3-kinase) activity is thought to play a role in trafficking from endosomes to lysosomes, wortmannin was used, by co-exposing cells to this kinase inhibitor and recombinant bFGF-SAP, presumably to avoid lysosomal degradation following endocytosis. When administered to mice, wortmannin combined with bFGF-SAP significantly decreased tumor volumes as compared to controls;147 however, when another kinase inhibitor, LY294002, was instead tested, it did not show the same effect. Thus, although wortmannin increased bGF-SAP cytotoxicity, potentiation may have occurred through an alternative pathway not involving Pl 3-kinase inhibition.

Brefeldin A (BFA) is a fungal metabolite known to inhibit ricin toxicity by disassembly of the Golgi apparatus,148, 149 and thereby affect ricin retrograde transport. BFA did not affect the toxicity of saporin or saporin-based immunotoxins,11 thus indicating a different localization for the latter inside Vero cells.

Paradoxic effects were also observed in a few cases for immunotoxins containing RTA and directed against surface receptors,150 or saporin-based immunotoxins against CD7 B cell marker (Ippoliti et al., unpublished results) where using sub-optimal concentrations of BFA (established as a dose which is not effective in blocking ricin subcellular delivery at short times of incubation) we could instead potentiate the activity of the CD7 immunotoxin even if the latter was also partly local-ized at the Golgi complex. These data further suggest that a simple accumulation at the level of Golgi stacks may not per se be sufficient to let these toxins reach the cytosol (by a TGN–ER pathway) indicating the possible involvement of other as yet unidentified routes in parallel, consis-tent with more recent reports.110 Saponins87 may allow the broadening of the therapeutic window with a simultaneous dose-lowering in chimeric toxin, since saporin chimeras can retain target spec-ificity. The saponin-mediated enhancement of saporin cytotoxicity was shown to be diminished by drugs interfering with clathrin-mediated endocytosis. On the other hand, inhibitors of caveolae-mediated endocytosis and drugs known to interfere with endosomal trafficking had no effect.151

Photochemical Internalization (PCI) of Toxins

A recent premeabilization technique makes use of photosensitizing agents that can preferentially localize in endocytic vesicles. The PCI method is based on the use of compounds, such as alu-minium phthalocyanine (AlPcS2a), which upon light exposure induce photochemical damage to the vesicular membranes, releasing intact macromolecules into the cytosol.152

By inducing endosomal content release upon light exposure,153, 154 PCI is a potent system to enhance the delivery of genes and several drugs or toxins within cancer cells. Gelonin toxicity was demonstrated155 to be greatly enhanced by the photochemical re-localization of the photosensitizer AlPcS2a. Fluorescently-labeled gelonin was co-localized inside endosomes and lysosomes together with AlPcS2a. Upon light exposure, both the toxin and sensitizer were found to be completely dif-fusing in the cytosol. A similar effect was observed with EGF–biotin–streptavidin–saporin156 in


EGF-R positive cells, where the photoactivation of TPPS2a compound nicely increased the toxicity of this targeted toxin. The authors could also demonstrate an increased cytotoxicity of unconjugated saporin, but not of a streptavidin-conjugated saporin, suggesting streptadavin may alter the locali-zation of the uncojugated toxin. In fact a biotin–streptavidin linkage to saporin was also used for obtaining the murine-human IgG1 monoclonal Cetuximab (also termed C225 or “Erbitux”) chi-meric toxin, where PCI again enhanced the cytotoxicity of the latter immunotoxin by favoring its release from the endo-lysosomal compartments.157

Recently,158 Selbo and co-workers used PCI to enhance ScFv-based gelonin chimeras directed against melanoma cells and a polyamidoamine (PAMAM) dendrimer-delivered saporin chimera.159 In this latter study, saporin was reported to accumulate in the cytosol of the light-exposed cells but, surprisingly, also inside the nucleus, a phenomenon that could be linked to the presence of the dendrimer.

All the above reported studies included controls of the PCI effect on cells not exposed to the toxins, thus suggesting that accumulation of the RIP inside endosomal and lysosomal vesicles is presumably the only reason for the potentiation induced by the photosensitizers. Effects of PCI on other type 1 RIPs should be investigated.

Conclusions and Perspectives

The exploitation of type 1 RIPs for cancer therapy has elicited a deeper investigation into their structural and biophysical properties. Based on our current knowledge, evolution has diverged be tween type 1 and type 2 A chains, possibly facilitating the latter to better adapt their sequence to the intracellular environment reached within the ER. As flexible/unfoldable proteins facilitated by partner proteins, the B binding domains trigger efficient endocytosis. Type 1 RIPs would seem to have limited their intracellular journey to the cytosol to other endocytic compartments. One possi-bility being that they are taking advantage of their greater stability to escape enzymatic degradation in lytic compartments having evolved not yet fully understood underlying mechanism(s) to gain access to the cytoplasm.

Both RIP classes represent useful tools for cell biologists to dissect endocytotic pathways, with N-glycosidase activity being a “user-friendly” method to assess internalization/cytosolic delivery. From this perspective, we believe RIPs will continue to interest researchers in different fields for a long time to come.

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