Báo cáo khoa học: Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate doc

Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate Sandra E. Geden 1 , Richard A. Gardner 2 , M. Serena Fabbrini 3 , Masato Ohashi 4 , Otto Phanstiel IV 2 and Ken Teter 1 1 Department of Molecular Biology and Microbiology and Biomolecular Science Center, University of Central Florida, FL, USA 2 Department of Chemistry and Biomolecular Science Center, University of Central Florida, FL, USA 3 Istituto Biologia e Biotecnologia Agraria, CNR, Milan, Italy 4 Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, Japan Saporin is a lethal 30 kDa ribosome-inactivating protein from the plant Saponaria officinalis [1]. The toxin lacks an efficient cell-binding moiety and therefore exhibits low in vivo activity. However, it is possible to append saporin with a specific cell-binding motif. Urokinase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin [2]. The N-terminal domain of uPA specifically targets the toxin conjugate to uPA receptors (uPARs) that are highly expressed in human breast, colon, and prostate cancers [2–5]. uPA-saporin can thus selectively target and kill Keywords anticancer therapy; endosome; intracellular trafficking; plant ribosome-inactivating protein; polyamine Correspondence K. Teter, Biomolecular Research Annex, 12722 Research Parkway, Orlando, FL 32826, USA Fax: +1 407 384 2062 Tel: +1 407 882 2247 E-mail: kteter@mail.ucf.edu (Received 12 June 2007, revised 20 July 2007, accepted 23 July 2007) doi:10.1111/j.1742-4658.2007.06008.x Saporin is a type I ribosome-inactivating protein that is often appended with a cell-binding domain to specifically target and kill cancer cells. Uroki- nase plasminogen activator (uPA)-saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin enter the target cell by endo- cytosis and must then escape the endomembrane system to reach the cyto- solic ribosomes. The latter process may represent a rate-limiting step for intoxication and would therefore directly affect toxin potency. In the pres- ent study, we document two treatments (shock with dimethylsulfoxide and lipopolyamine coadministration) that generate substantial cellular sensitiza- tion to saporin ⁄ uPA-saporin. With the use of lysosome-endosome X (LEX)1 and LEX2 mutant cell lines, an endosomal trafficking step preced- ing cargo delivery to the late endosomes was identified as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide and lipopolyamines are known to disrupt the integrity of endosome membranes, so these reagents could facilitate the rapid movement of toxin from permeabilized endosomes to the cytosol. However, the same pattern of toxin sensitization was not observed for dimethylsulfoxide- or lipopolyamine-treated cells exposed to diphtheria toxin, ricin, or the cata- lytic A chain of ricin. The sensitization effects were thus specific for saporin, suggesting a novel mechanism of saporin translocation by endosome disrup- tion. Lipopolyamines have been developed as in vivo gene therapy vectors; thus, lipopolyamine coadministration with uPA-saporin or other saporin conjugates could represent a new approach for anticancer toxin treatments. Abbreviations CHO, Chinese hamster ovary; DT, diphtheria toxin; EC 50 , half-maximal effective concentration; ER, endoplasmic reticulum; ERAD, ER-associated degradation; LEX, lysosome-endosome X; LT IIb, Escherichia coli heat-labile toxin IIb; uPA, urokinase plasminogen activator; uPAR, uPA receptor. FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS 4825 these cancer cells. Other saporin conjugates, chimeras, and immunotoxins have also been developed as antican- cer agents [6]. After binding to the cell surface, saporin and anti- cancer saporin conjugates must enter the cytosol to inactivate the ribosomes. This event may represent a rate-limiting step for the intoxication process and could therefore directly affect toxin potency. Efficient toxin delivery to the cancer cell cytosol would accord- ingly enhance the therapeutic value of anticancer sapo- rin conjugates. Unfortunately, saporin passage into the cytosol remains a poorly understood process. Both saporin and anticancer saporin conjugates enter the target cell by endocytosis and then escape the endomembrane system to reach their cytosolic target [7–11]. Other toxins that follow this general pathway exit the endomembrane system from either acidified endosomes or the endoplasmic reticulum (ER) [12]. These toxins generally exhibit an AB structural organi- zation that consists of an enzymatic A subunit and a cell-binding B subunit. AB toxins that move from the endosomes to the cytosol undergo an acid-dependent conformational change in the translocation domain that creates a pore into the endosome membrane, which permits passage of the toxin catalytic subunit into the cytosol. A second category of AB toxins trav- els from the endosomes to the Golgi apparatus en route to an ER translocation site. These ER-translo- cating toxins pass through the Sec61 translocon, a pre- existing pore in the ER membrane, in order to move from the ER to the cytosol. For both endosome and ER exit sites, A ⁄ B subunit dissociation and A chain unfolding occur before or during toxin export. The site of endomembrane escape for saporin, which can be viewed as a toxin A chain without the corresponding B subunit, remains uncertain because its intracellular transport route bypasses the Golgi apparatus [9]. In this work, our studies to delineate the route of sa- porin entry into the cytosol have identified two experi- mental conditions that generate significant cellular sensitization to saporin. Both reagents used for these conditions (dimethylsulfoxide or polycationic lipopoly- amines) can disrupt the integrity of endosome mem- branes [13,14]. Neither reagent generated comparable levels of cellular sensitization to AB toxins or to the isolated A chain of the plant toxin ricin. Thus, there appeared to be a synergistic effect between saporin and dimethylsulfoxide or lipopolyamine, which resulted in permeabilization of the endosome mem- brane and direct delivery of the toxin to the cytosol. Additional studies identified an endosomal trafficking step preceding cargo delivery to the late endosomes as a major site for the dimethylsulfoxide-facilitated entry of saporin into the cytosol. Dimethylsulfoxide- or lipopolyamine-induced toxin sensitization was also observed in a model cancer cell line exposed to the anticancer toxin uPA-saporin. Because lipopolyamines have been developed as nontoxic in vivo gene therapy vectors [14–16], lipopolyamine coadministration with uPA-saporin or other saporin conjugates could repre- sent a new approach for anticancer toxin treatments. Results and Discussion Saporin intoxication does not require the mechanism of ER-associated degradation Expression of an anticancer saporin chimera in the ER of Xenopus oocytes produced a drastic inhibition of translation in the RNA-injected cells [17]. Injection of neutralizing saporin antibodies in the host cell cytosol abolished the toxic effect, indicating that some of the newly synthesized chimeric toxin mislocalized into the cytosol. Most toxins that reach the ER exploit the quality control system of ER-associated degradation (ERAD) for translocation to the cytosol [18,19]. An ER-to-cytosol export of this saporin chimera could, in principle, also result from ERAD activity. This would be consistent with the observation that the 3D struc- ture of saporin can be superimposed with the A chain of ricin, an ER-translocating toxin [20]. Yet, even cor- rectly folded proteins are exported from the ER to the cytosol to some small extent. If this occurred for the saporin chimera, it could affect translation, even though only a small pool of newly synthesized toxin was mistakenly exported to the oocyte cytosol. Alter- natively, the inhibition of translation could have resulted from inefficient toxin insertion into the ER. To detect a functional role for ERAD in intoxica- tion with exogenously applied saporin, we generated toxin dose–response curves for wild-type and mutant Chinese hamster ovary (CHO) cells (Fig. 1). The mutant cell lines exhibit defects in the ERAD pathway that generate substantial cellular resistance to a num- ber of established ER-translocating toxins [21,22]. Accelerated ERAD activity in mutant clones 23 and 24 [22] conferred resistance to Escherichia coli heat-labile toxin IIb (LT IIb; Fig. 1A), a known ER- translocating toxin, but did not inhibit intoxication with saporin (Fig. 1B). The disruption of ERAD-medi- ated toxin translocation in mutant clones 16 and 46 [21] also conferred resistance to LT IIb (Fig. 1C) but, again, no inhibitory effect on saporin could be detected (Fig. 1D). Because at least two distinct defects in the ERAD system failed to generate resistance against saporin, this toxin would appear to utilize an Toxin translocation by endosome disruption S. E. Geden et al. 4826 FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS ERAD-independent mechanism of translocation across the ER membrane. Alternatively, translocation of exogenous saporin into the cytosol may have occurred from an organelle other than the ER. Internalized saporin and uPA-saporin have not been detected in the Golgi apparatus or the ER [9]. Further- more, saporin intoxication is not affected by the inhibi- tion of ER-to-Golgi ⁄ Golgi-to-ER vesicular trafficking that is elicited by treatment with the fungal drug brefel- din A [9]. Intoxication with several other ER-translo- cating toxins is effectively blocked by brefeldin A [18]. Finally, saporin does not follow the intracellular trans- port routes utilized by ricin holotoxin and ricin A chain to enter the host cell cytosol from the ER [9]. These observations, combined with our results from the ERAD-defective CHO mutants, suggested that saporin translocation may actually occur from the endosomes. A postintoxication shock with dimethylsulfoxide generates significant cellular sensitization to saporin Exit of saporin from the endomembrane system would likely occur by a novel mechanism, different from the established pathway described for diphtheria toxin (DT). Toxins such as DT that move from the endo- somes to the cytosol rely upon an acid-induced confor- mational change in a toxin subunit to facilitate export. Endosome alkalinization thus generates resistance to toxins that move from the endosomes to the cytosol. However, treatment with chloroquine or bafilomy- cin A1 (two drugs that alkalinize the endosomal com- partments) does not inhibit saporin intoxication [9]. The putative endosome-to-cytosol export of saporin would therefore be distinct from the mechanism uti- lized by other toxins that follow this translocation route. The DEAE-dextran transfection method, a procedure that involves disruption of the endosomal membrane, represented an alternative pathway for entering the cytosol from the endosomes. The DEAE-dextran com- plex is a polycationic reagent that, by virtue of its physi- cal properties, disrupts the endosomal membrane and thereby allows DNA to enter the cytosol [15,23]. Details of the mechanism by which endosome disruption occurs is still a matter of debate, but recent lipopolyamine DNA transfection protocols are thought to operate by a similar mechanism of endosomal disruption [14]. CD AB Fig. 1. Effect of ERAD dysfunction on sapo- rin intoxication. Cellular sensitivity to LT IIb or saporin was monitored in wild-type CHO cells and mutant CHO cells with aberrant ERAD activity. (A,B) Intoxication with LT IIb (A) or saporin (B) was monitored in mutant cell lines with accelerated ERAD activity (clones 23 and 24). (C,D) Intoxication with LT IIb (C) or saporin (D) was monitored in mutant cell lines with attenuated ERAD- mediated translocation (clones 16 and 46). For LT IIb intoxication assays, cells were incubated with varying concentrations of toxin for 2 h before intracellular cAMP levels were quantified with a [ 125 I]cAMP competi- tion assay. LT IIb is an ER-translocating toxin that stimulates cAMP production in the target cell. The mean ± SEM is shown for four independent experiments with tripli- cate samples. For saporin intoxication assays, cells incubated with varying concen- trations of toxin for 8 h were chased in toxin-free media for 16 h before protein syn- thesis levels were quantified. The mean ± SEM is shown for at least three indepen- dent experiments with triplicate samples. S. E. Geden et al. Toxin translocation by endosome disruption FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS 4827 Saporin has an extremely high pI (approximately 10) and thus bears some biochemical similarity to DEAE- dextran (pI ¼ 10.8) and lipopolyamine (pI > 9) trans- fection reagents. As such, we hypothesized that saporin enters the cytosol by disrupting an endosome mem- brane. Such a process would not require the unfolding of saporin, which indeed appears to be an extremely stable protein [24]. The low level of toxicity obtained with exogenously applied saporin suggests that this putative endosome disruption mechanism is a low fre- quency event. DEAE-dextran tranfections are also low frequency events, but the efficiency of transfection is greatly enhanced by a 2-min shock with dimethylsulf- oxide at the end of the DEAE-dextran exposure [25]. Dimethylsulfoxide shock provides an additional, effi- cient mechanism for endosome permeabilization [13]. Thus, we reasoned that a dimethylsulfoxide shock would enhance the potency of saporin. We exposed cells to saporin for 8 h and then, as per the DEAE-dextran transfection method, initiated a 2-min shock with 10% dimethylsulfoxide in toxin-free media (Fig. 2). The cells were then incubated for another 16 h in the absence of saporin and dimethylsulfoxide before determining the extent of intoxication. As shown in Fig. 2A, cells shocked with dimethylsulfoxide were 125-fold more sensitive to saporin at the half-maximal effective concentration (EC 50 ) than cells that did not receive the shock treat- ment. A 2-min dimethylsulfoxide shock before saporin intoxication did not result in substantial toxin sensiti- zation (Fig. 2B); thus, the dramatic sensitization from a post-toxin dimethylsulfoxide shock cannot result from a general negative effect of dimethylsulfoxide on cell health. Indeed, the dimethylsulfoxide shock treat- ment only reduced protein synthesis levels in unintoxi- cated cells to 80% of the untreated, unintoxicated control level. Dimethylsulfoxide treatment may have transiently permeabilized the cell membrane and allowed any remaining residual toxin to directly enter the cytosol. To control for this possibility, we (a) placed cells in saporin-containing media; (b) immedi- ately removed the media; (c) shocked the cells for 2 min with dimethylsulfoxide in toxin-free media; (d) returned the saporin-containing media to the cells for 8 h; and (e) chased the cells overnight in the absence of toxin. This procedure, in which the dimethylsulfox- ide shock precedes the 8 h toxin incubation but occurs after a very brief exposure to saporin, did not result in substantial sensitization to saporin (Fig. 2B). Signifi- cant toxin sensitization was only seen when the dimethylsulfoxide shock occurred after an 8 h toxin incubation permitted saporin endocytosis (Fig. 2A). Dimethylsulfoxide treatment thus appeared to specifi- cally affect the endocytosed pool of saporin. Endocytic trafficking is involved with the dimethylsulfoxide-induced sensitization to saporin To confirm that endocytosis was required for the dimethylsulfoxide sensitization effect, we repeated our postintoxication shock protocol with cells exposed to saporin for 4 h at 4 °C (Fig. 3A). Endocytosis is effec- tively blocked at 4 °C. Saporin intoxication was also blocked at this temperature, as exposure of up to 25 lg saporinÆ mL )1 at 4 °C was not sufficient to A B Fig. 2. Effect of dimethylsulfoxide (DMSO) on saporin intoxication. CHO cells were incubated for 8 h with varying concentrations of saporin. The cells were then chased in toxin-free media for 16 h before protein synthesis levels were monitored. Results were expressed as percent- ages of the values obtained from unintoxicated cells treated in an identical manner to the corresponding toxin-exposed cells. The mean ± SEM is shown for at least three independent experiments with triplicate samples. (A) A 2 min shock with 10% DMSO followed the toxin incubation (+ DMSO). (B) A 2 min shock with 10% DMSO preceded the toxin incubation (DMSO–toxin), or cells were placed in toxin- containing media; the media was removed; the cells were shocked for 2 min with 10% DMSO; toxin-containing media was returned to the cells for 8 h; and the cells were chased 16 h without toxin (toxin–DMSO–toxin). Toxin translocation by endosome disruption S. E. Geden et al. 4828 FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS produce an EC 50 value. In this assay, an overnight chase at 37 °C followed the 4 °C toxin exposure. Thus, the surface-bound toxin present at 4 °C was endocyto- sed during the 37 °C chase to generate a minor but productive intoxication. Some degree of toxin sensiti- zation was seen when cells were shocked with dimethyl- sulfoxide after exposure to saporin at 4 °C. However, the two-fold level of sensitization observed for this 4 °C condition (compared to the 37 °C intoxication) was similar to the three-fold level of sensitization doc- umented for cells shocked with dimethylsulfoxide before toxin exposure (Fig. 2B). This indicated that a nonspecific dimethylsulfoxide effect was responsible for toxin sensitization when cells were exposed to saporin at 4 °C. The two-fold level of saporin sensiti- zation for dimethylsulfoxide-treated cells incubated with toxin at 4 °C was clearly attenuated in compari- son to the 125-fold level of saporin sensitization for dimethylsulfoxide-treated cells incubated with toxin at 37 °C (Fig. 2A). Endocytosis was therefore required for the dramatic dimethylsulfoxide-induced sensiti- zation to saporin. Dimethylsulfoxide-induced saporin sensitization was also examined in the lysosome-endosome X (LEX)1 and LEX2 mutant cell lines (Fig. 3B,C). The LEX1 mutant is defective in transport from the late endo- somes to the lysosomes, whereas the LEX2 mutant accumulates multivesicular bodies that serve as (a) sort- ing stations and (b) transport intermediates between the early endosomes and late endosomes [26,27]. LEX1 cells were more sensitive to saporin than the parental cells from which the LEX1 and LEX2 mutants were derived (Fig. 3B). However, the LEX1 and parental cells produced identical toxin dose–response curves after dimethylsulfoxide shock (Fig. 3B). By contrast, the LEX2 cells were more sensitive to saporin intoxica- tion than the parental cells both with and without a dimethylsulfoxide shock (Fig. 3C). The LEX2 and LEX1 cells exhibited similar levels of saporin sensitivity in the absence of dimethylsulfoxide shock. Collectively, our results suggest an endosomal traf- ficking step preceding cargo delivery to the late endo- somes is the major site for dimethylsulfoxide-facilitated entry of saporin into the cytosol. The LEX2 inhibition of cargo delivery to the late endosomes would thereby increase the pool of saporin available for passage from the early endosomes and ⁄ or multivesicular bodies to the cytosol. This, in turn, would generate an elevated state of dimethylsulfoxide-induced toxin sensitivity in the LEX2 cells relative to the parental and LEX1 cells. Selective permeabilization of the early endosomes and ⁄ or multivesicular bodies by dimethylsulfoxide treatment would explain why toxin accumulated in the late endosomes of LEX1 cells did not generate a simi- lar elevated state of dimethylsulfoxide-induced toxin sensitivity. Because both LEX2 and LEX1 cells were more sensitive to saporin than the parental cells in the absence of dimethylsulfoxide shock, the late ABC Fig. 3. Role of endocytic trafficking in dimethylsulfoxide (DMSO)-induced sensitization to saporin. (A) CHO cells were preincubated for 30 min at 4 °C in serum-free medium buffered with 20 m M Hepes pH 7.2. The cells were then incubated in Hepes-buffered medium with varying concentrations of saporin for an additional 4 h at 4 °C. For one set of cells, a 2 min 37 °C shock with 10% DMSO followed the toxin incubation (4 °C + DMSO). The cells were then chased in toxin-free media for 20 h before protein synthesis levels were monitored. A third set of cells were kept at 37 °C for the entire experiment, including the 4 h toxin exposure. (B,C) Varying concentrations of saporin were added at 37 °C for 4 h to LEX1 cells (B), LEX2 cells (C), and the parental cells from which the LEX mutants were derived. For one set of each cell type, a 2 min shock with 10% DMSO followed the toxin incubation (+ DMSO). The cells were then chased in toxin-free media for 20 h before protein synthesis levels were monitored. Data in (B) and (C) were generated simultaneously but are presented separately for clarity. For all experiments, results were expressed as percentages of the values obtained from unintoxicated cells treated in an identical manner to the corresponding toxin-exposed cells. The mean ± SEM is shown for least four independent experiments with triplicate samples. S. E. Geden et al. Toxin translocation by endosome disruption FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS 4829 endosomes may also serve as a saporin translocation site. The inhibition of cargo delivery to the lysosomes in either LEX2 or LEX1 cells would again increase the pool of saporin available for passage from the endo- somes to the cytosol and would thereby generate an elevated state of toxin sensitivity in the LEX mutants. Escape from the endomembrane system is a possible rate-limiting step in saporin intoxication We also performed a dimethylsulfoxide shock on CHO cells that had only been exposed to saporin for 1 h (Table 1). This procedure resulted in an EC 50 of 1 lgÆmL )1 after just 3 h of chase. Without a dimethylsulfoxide shock, the effects of intoxication were observed 24 h after the initial toxin exposure but not at 4 h postexposure. Furthermore, with an over- night chase in the absence of dimethylsulfoxide shock, a 1 h toxin exposure was only slightly less effective than the 4 h and 8 h toxin exposures. These data indi- cated that saporin can enter the endomembrane system relatively quickly (within an hour), but its exit from the endomembrane system and the corresponding man- ifestation of toxicity occur slowly. Saporin escape from the endomembrane system thus appeared to represent a rate-limiting step during the intoxication process. Dimethylsulfoxide treatment accelerated the rate and extent of saporin entry into the cytosol, which in turn greatly enhanced toxin potency. Dimethylsulfoxide treatment does not induce substantial sensitization to other toxins To determine the specificity of dimethylsulfoxide- induced toxin sensitization, we repeated our experi- ment with cells exposed to AB toxins with established endosome (i.e. DT) or ER (i.e. ricin) exit sites (Fig. 4). A dimethylsulfoxide shock had no effect on DT Table 1. Time course of saporin intoxication. CHO cells were exposed to varying concentrations of saporin for 1–8 h. Inhibition of protein synthesis was then quantified after a total time interval (toxin exposure + chase) of 4 or 24 h. EC 50 values were calculated from four independent experiments with triplicate samples. Toxin exposure EC 50 (lgÆmL )1 ) 4 h 24 h 1h >25 a 8 4h >25 a 2.5 8h – 2 1 h + dimethylsulfoxide shock 1 – a Cells treated with 25 lg saporinÆmL )1 exhibited approximately 85% of the protein synthesis levels recorded for unintoxicated con- trol cells. AB CD Fig. 4. Effect of dimethylsulfoxide (DMSO) on intoxication with diphtheria toxin, ricin, or ricin A chain. CHO cells were incubated for 4 h with varying concentrations of DT (A), ricin (B), or ricin A chain (C). In (D), LEX2 cells and their parental cells were incubated for 4 h with varying concentrations of ricin A chain. Where indicated, a 2 min shock with 10% DMSO followed the toxin incuba- tion (+ DMSO). The cells were then chased in toxin-free media for 20 h before protein synthesis levels were monitored. Results were expressed as percentages of the values obtained from unintoxicated cells treated in an identical manner to the corresponding toxin-exposed cells. The mean ± SEM is shown for at least three independent experiments with triplicate samples. Toxin translocation by endosome disruption S. E. Geden et al. 4830 FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS (Fig. 4A) and only resulted in a 2.5-fold level of sensi- tization to ricin (Fig. 4B). This low level of sensitiza- tion was also seen for cells that were shocked with dimethylsulfoxide before the addition of saporin and for cells shocked with dimethylsulfoxide after a 4 °C toxin incubation (Figs 2B and 3A). Sandvig et al. [28] have further shown that cells coincubated with dimethylsulfoxide and either DT or ricin for prolonged incubations are actually more resistant to these toxins than cells incubated without dimethylsulfoxide. Collec- tively, our results demonstrated that dimethylsulfoxide does not elicit a general sensitization to AB toxins with established endosome or ER exit sites. It was also possible that dimethylsulfoxide alone was responsible for membrane disruption and that saporin played no active role in the process. With this scenario, DT and ricin might not be more toxic after a dimethylsulfoxide shock because they would enter the cell as intact AB toxins and not as isolated, catalyti- cally active A chains. To control for this possibility, we repeated the dimethylsulfoxide shock experiment with cells exposed to free ricin A chain (Fig. 4C). If dimethylsulfoxide alone permeabilized the endosome membrane, then the dimethylsulfoxide shock would increase the potency of ricin A chain because the toxin would move directly from the endosomes to the cyto- sol. This could occur even though ricin A chain nor- mally moves from the endosomes to the ER before translocation into the cytosol [9,29]. We indeed detected a ten-fold increase in cellular sensitivity to ricin A chain after the dimethylsulfoxide shock, as would be expected from A chain escape during transit through the endosome compartments. However, the relative effect of sensitization to ricin A chain was sub- stantially lower than the 125-fold level of dimethylsulf- oxide-induced sensitization to saporin. In the absence of dimethylsulfoxide treatment, saporin was only three-fold more toxic than ricin A chain at the EC 50 value (Figs 2 and 4C), so the large differences between saporin and ricin A chain sensitization were unlikely to reflect possible differences in the intrinsic toxin activities. Furthermore, our evaluations of toxin sensi- tization involved internal standard controls: saporin intoxication with dimethylsulfoxide shock was expressed as a relative value of saporin intoxication without dimethylsulfoxide shock, whereas ricin A chain intoxication with dimethylsulfoxide shock was expressed as a relative value of ricin A chain intoxica- tion without dimethylsulfoxide shock. The dramatic increase in cellular sensitivity to saporin after a dimethylsulfoxide shock thus appeared to result from a synergistic toxin ⁄ dimethylsulfoxide effect that was more profound with saporin. We found that the elevated state of dimethylsulfox- ide-induced toxin sensitivity in the LEX2 cells also dif- fered for saporin and ricin A chain. By contrast to the dimethylsulfoxide ⁄ saporin experiment (Fig. 3C), LEX2 cells and their parental cells produced identical dose– response curves for a dimethylsulfoxide⁄ ricin A chain experiment (Fig. 4D). The LEX2 and parental cells also exhibited similar sensitivities to ricin A chain in the absence of dimethylsulfoxide shock (Fig. 4D), which was again distinct from the results of the saporin experi- ment (Fig. 3C). Saporin and ricin A chain normally follow separate pathways into the cytosol [9,29]; these results demonstrated that the two toxins also follow separate trafficking ⁄ translocation itineraries into the cytosol after a dimethylsulfoxide shock. Thus, dimethyl- sulfoxide treatment appeared to specifically enhance the productive pathway of saporin intoxication. Lipopolyamine treatment enhances saporin intoxication DNA transfections with lipopolyamines can effectively disrupt the endosome membrane without a dimethyl- sulfoxide shock [14]. Thus, similar to the dimethylsulf- oxide shock, coadministration of lipopolyamines with saporin should generate significant cellular sensitization to the toxin. We tested this prediction using three lipo- polyamine vectors with different relative DNA transfec- tion efficiencies [16]. These lipopolyamines are shown in Figure 5; they differ in the number of evenly spaced positive charges along the polyamine scaffold. O O O O O O N H 2 NH 3 + + 2Cl _ 1 N H 2 H 2 N NH 3 + + + 3Cl _ 2 N H 2 H 2 N N H 2 NH 3 + + + + 4Cl _ 3 Fig. 5. Lipopolyamine structures. The molecular weights of 1–3, as previously described [16], are 784, 891, and 999 gÆmol )1 , respectively. S. E. Geden et al. Toxin translocation by endosome disruption FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS 4831 Cells coincubated with saporin and 5 lgÆmL )1 of lipo- polyamine were between 33- and 83-fold more sensitive to saporin at the EC 50 value than cells incubated with saporin in the absence of lipopolyamine (Table 2). Lipopolyamine treatment alone reduced protein synthe- sis to either 60% (lipopolyamines 1 and 2) or 80% (lipopolyamine 3) of the untreated, unintoxicated con- trol level. Internal controls accounted for this lipopoly- amine-induced effect on protein synthesis. Interestingly, the degree of saporin sensitization correlated directly with the transfection efficacy of the lipopolyamine. By contrast, the three tested lipopolyamines did not sensi- tize cells to DT (not shown) and generated only rela- tively modest levels of sensitization to ricin, with the exception of lipopolyamine 3, which actually conferred resistance to the ricin holotoxin. Sensitization to ricin A chain was detected, but there was an inverse relation- ship between the extent of lipopolyamine-induced sensi- tization to saporin versus free ricin A chain. Thus, as with the extent of dimethylsulfoxide-induced toxin sen- sitization and the susceptibility of LEX2 cells to intoxi- cation, significant differences were recorded for the cellular response to saporin versus ricin A chain. The distinct pattern of lipopolyamine-induced sensitization to ricin A chain could result from membrane destabili- zation by ricin A chain [30,31] and⁄ or lipopolyamine disruption of endo-lysosomal compartments other than the early endosomes and multivesicular bodies [14,15]. Future studies will examine these possibilities and their implications for the delivery of immunotoxins contain- ing ricin A chain. Here, a specific sensitization effect that directly corresponded to lipopolyamine transfec- tion efficiency was documented for saporin intoxication. Lipopolyamine treatment enhances the potency of uPA-saporin, an anticancer toxin conjugate Lipopolyamines are being developed as nontoxic in vivo gene therapy vectors [14–16]. These compounds could accordingly be used as therapeutic agents to increase the efficiency of cell killing by saporin-based anticancer toxins. To examine this possibility in tissue culture cells, we examined whether lipopolyamine 3 could sensitize a model cancer cell line to uPA-sapo- rin (Fig. 6). LB6 murine fibroblasts and LB6 clone 19 cells that stably express the human uPAR were used for this purpose [32]. Because many human cancer cells overexpress the uPAR [3–5], LB6 clone 19 can serve as a model cancer cell line to assess the efficacy of anticancer toxin treatments [2,7,8]. Both LB6 and LB6 clone 19 cells were used to distinguish general cytotoxic effects from uPAR ⁄ cancer-specific cell kill- ing. Lipopolyamine 3 was chosen for this work because it was the most effective and least toxic of the three lipopolyamines tested on CHO cells (Table 2). We first confirmed that LB6 and LB6 clone 19 cells could be sensitized to native saporin by a dimethyl- sulfoxide shock and by lipopolyamine treatment (Fig. 6A,B). For these cells, the dimethylsulfoxide shock reduced protein synthesis levels to 60% of the untreated, unintoxicated control level. Treatment with lipopolyamine 3 had no adverse effect on protein syn- thesis. In our experimental conditions, native saporin exhibited minimal toxicity against the LB6 (Fig. 6A) and LB6 clone 19 cells (Fig. 6B). However, toxicity was observed when the saporin-treated cells were sub- jected to a dimethylsulfoxide shock and when lipopoly- amine 3 was coadministered with saporin. The dimethylsulfoxide shock generated substantially greater sensitization to saporin than lipopolyamine treatment for both LB6 and LB6 clone 19 cells. In comparison, dimethylsulfoxide-treated CHO cells were only slightly more sensitive to saporin than lipopolyamine-treated CHO cells (Fig. 2A and Table 2). CHO cells were also susceptible to saporin intoxication in the absence of additional treatment (Fig. 2). Collectively, these obser- vations suggested that the endosomes of LB6 and LB6 Table 2. Lipopolyamine-induced toxin sensitization. 5 lgÆmL )1 of the stated lipopolyamine was administered to CHO cells with varying con- centrations of toxin for a 4 h coincubation. EC 50 values from cells incubated in the absence or presence of lipopolyamine were then deter- mined at 24 h postintoxication. Values were calculated from protein synthesis levels with 3–5 experiments per condition. The levels of lipopolyamine-induced toxin sensitization (in comparison to toxin-treated cells incubated without lipopolyamine) are shown. Lipopolyamine Transfection efficiency a Saporin sensitization Ricin sensitization Ricin A chain sensitization Compound 1 + 33-fold 8-fold 250-fold Compound 2 ++ 63-fold 15-fold 200-fold Compound 3 +++ 83-fold None b 13-fold a Relative transfection efficiency as previously reported [16]. b Cells exposed to compound 3 were highly resistant to ricin and exhibited, at a toxin concentration of 10 ngÆmL )1 , 64% of the protein synthesis levels recorded for the unintoxicated control cells. In comparison, cells incu- bated with 10 ng ricinÆmL )1 in the absence of lipopolyamine exhibited 22% of the protein synthesis levels recorded for unintoxicated control cells. Toxin translocation by endosome disruption S. E. Geden et al. 4832 FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS clone 19 cells were more resistant to saporin ⁄ lipopoly- amine membrane disruption than CHO cells. The extent of lipopolyamine-induced toxin sensitization is thus likely to vary amongst different cell types and may depend upon endosome membrane composition. We next determined whether LB6 and LB6 clone 19 cells could be sensitized to uPA-saporin by a dimethylsulfoxide shock or lipopolyamine treatment (Fig. 6C,D). As expected, uPA-saporin exhibited very little toxicity against the parental LB6 cells, although dimethylsulfoxide shock or lipopolyamine coadminis- tration did result in some sensitization to the toxin (Fig. 6C). LB6 clone 19 cells were susceptible to uPA- saporin intoxication and were further sensitized to the toxin by dimethylsulfoxide shock or lipopolyamine coadministration (Fig. 6D). Dimethylsulfoxide- and lipopolyamine-induced sensitization to uPA-saporin were both several-fold higher in the LB6 clone 19 cells than in the parental LB6 cells, indicating the specificity of the sensitization effect for cells that express the human uPAR. In the LB6 clone 19 cells, dimethylsulf- oxide was more effective at uPA-saporin sensitization than lipopolyamine coadministration. However, the latter condition still produced an approximate ten-fold level of toxin sensitization compared to cells incubated with uPA-saporin in the absence of additional treat- ment. Similar results were obtained after continuous exposure of the LB6 clone 19 cells to uPA-saporin or a uPA-saporin ⁄ lipopolyamine 3 mixture for 24 h (data not shown). The similar EC 50 values for uPA-sapo- rin ⁄ lipopolyamine 3 treatment after 4 h and 24 h of toxin exposure again indicated that toxin escape from the endomembrane system, rather than toxin endocyto- sis, was the rate-limiting step for intoxication in these experiments. Conclusions Dimethylsulfoxide shock or lipopolyamine treatment greatly enhances the potency of endocytosed saporin and uPA-saporin. The molecular basis for dimethyl- sulfoxide- or lipopolyamine-induced toxin sensitization remains to be elucidated, although the mechanism most likely involves disruption of the endosome membrane. Endosome disruption by the process of photochemical internalization [33,34] or saponin administration [35,36] has also been developed as a method to introduce various type I ribosome inacti- vating plant toxins into the target cell cytosol. The present study suggests that endosome disruption can AB CD Fig. 6. Effect of lipopolyamine treatment on uPA-saporin intoxication. LB6 cells (A,C) and LB6 clone 19 cells (B,D) were incubated for 4 h with varying concentrations of native saporin (A,B) or uPA-saporin (C,D). The cells were then chased in toxin-free media for 20 h before protein synthesis levels were monitored. Results were expressed as per- centages of the values obtained from un- intoxicated cells treated in an identical manner to the corresponding toxin-exposed cells. The mean ± SEM is shown for three independent experiments with triplicate samples. Circles represent untreated, toxin- exposed cells; triangles represent cells coin- cubated with toxin and lipopolyamine 3; squares represent cells that were subjected to a dimethylsulfoxide shock following toxin exposure. S. E. Geden et al. Toxin translocation by endosome disruption FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS 4833 successfully deliver these toxins to the cytosol because of their particular intracellular trafficking and ⁄ or translocation mechanisms. Because lipopolyamines have been designed as in vivo drug delivery vehicles, lipopolyamine coadmin- istration may represent a novel mechanism to increase the in vivo efficiency of cancer cell killing by saporin- based toxins. Short-term exposures to saporin ⁄ lipo- polyamine mixtures could be sufficient for treatment, given that a maximal increase in cell killing was achieved within the first four hours of LB6 clone 19 exposure to uPA-saporin ⁄ lipopolyamine 3. Future studies will determine the in vivo efficacy of lipopoly- amine-induced sensitization to saporin-based toxins. Experimental procedures Materials Recombinant saporin isoform SAP3 [7] or seed-extracted saporin from Sigma-Aldrich (St Louis, MO, USA) were used to equal effect in cell killing assays. The anticancer uPA-saporin conjugate, provided by U. Cavallaro, was generated by conjugating seed-extracted saporin to an N-succinimidyl-3-(2-pyridyldithio)propionate-derivatized pro- uPA as previously described [2]. LT IIb, provided by R. K. Holmes, was purified from E. coli strain HB101 transformed with the pTC100 expression plasmid [37]. Ricin A chain and ricin holotoxin were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA), whereas DT was purchased from Sigma-Aldrich. LB6 control cells and LB6 clone 19 cells that stably express the human uPAR [32] were a generous gift from F. Blasi. ERAD- defective CHO clones 16, 23, 24, and 46 were isolated from a screen which challenged mutagenized cells with a combi- nation of two lethal ER-translocating toxins, ricin and Pseudomonas aeruginosa exotoxin A [21,22]. The LEX1 and LEX2 cell lines were isolated by flow cytometry with a pro- cedure designed to identify mutagenized cells defective in the degradation of endocytosed low-density lipoprotein [26,27]. The synthesis of lipopolyamines 1–3 has been described previously [16]. Cell culture CHO cells (both our lab stock of CHO-K1 and the parental CHO cells for the LEX1 and LEX2 mutants), CHO mutants, the LEX1 mutant, and the LEX2 mutant were all grown under humidified conditions at 37 °C and 5% CO 2 in Ham’s F-12 media (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA) and penicillin ⁄ strep- tomycin (Gibco BRL). LB6 and LB6 clone 19 cells were grown under humidified conditions at 37 °C and 5% CO 2 in Dulbecco’s modified Eagle’s medium (Gibco BRL) sup- plemented with 10% fetal bovine serum and penicil- lin ⁄ streptomycin. DT, ricin, and saporin toxicity assays The toxin-mediated inhibition of protein synthesis was detected by [ 35 S]methionine (PerkinElmer Life And Analyti- cal Sciences, Inc., Waltham, MA, USA) incorporation into the newly synthesized proteins of toxin-treated cells. Cells were seeded at 50–80% confluency in 24-well plates and grown overnight. Serum-free medium containing varying concentrations of toxin was then added to the cells for 1, 4, 8, or 24 h. For incubations less than 24 h, the toxin-con- taining medium was removed and replaced with complete medium. The total duration of the intoxication (toxin expo- sure + chase) was 24 h unless otherwise noted. Where indicated, the cells were coincubated with toxin and 5 lg lipopolyamineÆmL )1 or were shocked with 10% dimethylsulfoxide in toxin-free, serum-free medium for 2 min before the chase was initiated. After intoxication, cells were placed in methionine-free medium for 30 min and were then exposed to approxi- mately 1 lCi [ 35 S]methionineÆmL )1 for 15 min. Radio- labeled cells were washed twice with ice-cold 10% trichloroacetic acid in NaCl ⁄ P i before solubilization in 0.2 N NaOH. [ 35 S]methionine incorporation into the newly synthesized, precipitated proteins of the cell extracts was determined by scintillation counting. Results from toxin- treated cells were expressed as percentages of the values obtained from control cells incubated without toxin. When additional treatments (i.e. dimethylsulfoxide shock or lipopolyamine administration) were performed on the intoxicated cells, a corresponding control condition (dimethylsulfoxide shock or lipopolyamine administration) was performed for the control cells incubated without toxin. Triplicate samples were used for all experiments. The following toxin concentrations were used for toxicity assays: DT, 1, 5, and 10 ngÆmL )1 ; ricin, 0.1, 0.5, 1, and ⁄ or 5ngÆmL )1 ; ricin A chain, 0.05, 0.1, 1, 5, 10, 25, and ⁄ or 50 lgÆmL )1 ; saporin, 0.01, 0.025, 0.05, 0.1, 1, 3, 5, 10, and ⁄ or 25 lgÆmL )1 ; uPA-saporin, 5, 25, 50, 250, 500, and ⁄ or 1000 ngÆmL )1 . The toxin dose which inhibited protein synthesis by 50% relative to the matched unintoxicated control cells was defined as EC 50 . These EC 50 values were obtained by plot- ting the average results from three to five independent experiments on a single toxin dose–response curve as explic- itly shown in Figs 1–4 and 6. Each independent experiment was conducted with three replicate wells per condition. For the data presented, an SEM of less than 10% was typically calculated for the averaged results of each toxin concentra- tion. Comparisons of toxin potency under various experi- mental conditions were made using the EC 50 values. Thus, a report of ‘n-fold sensitivity’ represents the relative change Toxin translocation by endosome disruption S. E. Geden et al. 4834 FEBS Journal 274 (2007) 4825–4836 ª 2007 The Authors Journal compilation ª 2007 FEBS [...]... condition within the experiment was set at 100%, and all other results were expressed as percentages of this value Acknowledgements We thank Dr Randall K Holmes (University of Colorado School of Medicine, CL, USA) for the gift of LT IIb; Drs Francesco Blasi and Massimo Resnati (Dibit-HSR, Milano, Italy) for the LB6 and clone 19 cells; Dr Ugo Cavallaro (IFOM, Milano, Italy) for the saporin conjugate; and. .. Benedetti PA, Brunori M & Citro G (1998) The effect of monensin and chloroquine on the endocytosis and toxicity of chimeric toxins Cell Mol Life Sci 54, 866–875 11 Cavallaro U & Soria MR (1995) Targeting plant toxins to the urokinase and alpha 2-macroglobulin receptors Semin Cancer Biol 6, 269–278 12 Sandvig K & van Deurs B (2002) Membrane traffic exploited by protein toxins Annu Rev Cell Dev Biol 18, 1–24 13... and Agnieszka Grabon for technical assistance This work was supported by start-up funds provided to K Teter from the University of Central Florida Department of Molecular Biology and Microbiology and an AIRC grant to M S Fabbrini Partial support was provided by the Broad Medical Research Program of the Eli and Edythe L Broad Foundation (O Phanstiel) Toxin translocation by endosome disruption References... plates and grown overnight Serum-free medium containing varying concentrations of toxin was then added to the cells for 2 h Intoxicated cells were subsequently solubilized in 0.75 mL of acidic ethanol (1 m HCl ⁄ EtOH in a 1 : 100 v ⁄ v ratio) for 15 min at 4 °C The supernatant was collected after a 10 min at 4 °C spin, and a second 4 °C extraction of the cell pellet was performed for 10 min with an ethanol... necessary to reach the EC50 value under the stated experimental condition Other studies with saporin and uPA -saporin have assessed toxicity after a 72 h experiment (48 h of toxin exposure with 24 h of chase) [2] However, in the present study, short incubation times (both toxin exposure and chase) were intentionally used to focus on dimethylsulfoxide- or lipopolyamine- induced sensitization effects The relatively... 23 Ogris M & Wagner E (2002) Tumor-targeted gene transfer with DNA polyplexes Somat Cell Mol Genet 27, 85–95 24 Santanche S, Bellelli A & Brunori M (1997) The unusual stability of saporin, a candidate for the synthesis of immunotoxins Biochem Biophys Res Commun 234, 129–132 25 Sussman DJ & Milman G (1984) Short-term, high-efficiency expression of transfected DNA Mol Cell Biol 4, 1641–1643 26 Ohashi... Sandvig K, Kirveliene V & Berg K (2000) Release of gelonin from endosomes and lysosomes to cytosol by photochemical internalization Biochim Biophys Acta 1475, 307–313 34 Yip WL, Weyergang A, Berg K, Tonnesen HH & Selbo PK (2007) Targeted delivery and enhanced cytotoxicity of cetuximab -saporin by photochemical internalization in EGFR-positive cancer cells Mol Pharm 4, 241–251 35 Bachran C, Sutherland... Supernatants from both extractions were combined and lyophilized cAMP levels in the lyophilized extracts were then quantified with an [125I]cAMP competition assay according to the manufacturer’s instructions (Amersham Biosciences, Piscataway, NJ, USA) cAMP levels from unintoxicated cells were also determined and were background subtracted from the experimental values The maximal cAMP value obtained under any... SK, Andersen JA & Andreasen PA (1996) Immunohistochemical localization of urokinase-type plasminogen activator, type-1 plasminogen-activator inhibitor, urokinase receptor and alpha(2)-macroglobulin receptor in human breast carcinomas Int J Cancer 66, 441–452 5 Verspaget HW, Sier CF, Ganesh S, Griffioen G & Lamers CB (1995) Prognostic value of plasminogen activators and their inhibitors in colorectal cancer... Carpani D, Soria MR & Fabbrini MS (2000) Endocytosis of a chimera between human pro-urokinase and the plant toxin saporin: an unusual internalization mechanism FASEB J 14, 1335–1344 9 Vago R, Marsden CJ, Lord JM, Ippoliti R, Flavell DJ, Flavell SU, Ceriotti A & Fabbrini MS (2005) Saporin and ricin A chain follow different intracellular routes to enter the cytosol of intoxicated cells FEBS J 272, 4983– . Lipopolyamine treatment increases the efficacy of intoxication with saporin and an anticancer saporin conjugate Sandra E. Geden 1 , Richard. (uPA) -saporin, for example, is an anticancer toxin that consists of a chemical conjugate between the human uPA and native saporin. Both saporin and uPA-saporin