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

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 r

intoxication with saporin and an anticancer saporin

conjugate

Sandra E Geden1, Richard A Gardner2, M Serena Fabbrini3, Masato Ohashi4, Otto Phanstiel IV2 and Ken Teter1

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 documpres-ent two treatmpres-ents (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; EC50, 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.

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, saposapo-rin 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

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]

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.

Saporin has an extremely high pI (approximately 10)

and thus bears some biochemical similarity to

DEAE-dextran (pI¼ 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

(EC50) 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 sensitisensiti-zation 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, unintoxiunintoxi-cated 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 4C (Fig 3A) Endocytosis is effec-tively blocked at 4C Saporin intoxication was also blocked at this temperature, as exposure of up to

25 lg saporinÆmL)1 at 4C was not sufficient to

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-toxin-containing media was returned to the cells for 8 h; and the cells were chased 16 h without toxin (toxin–DMSO–toxin).

produce an EC50 value In this assay, an overnight

chase at 37C followed the 4 C toxin exposure Thus,

the surface-bound toxin present at 4C was

endocyto-sed during the 37C 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 4C However,

the two-fold level of sensitization observed for this

4C 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 4C The two-fold level of saporin

sensiti-zation for dimethylsulfoxide-treated cells incubated

with toxin at 4C was clearly attenuated in

compari-son to the 125-fold level of saporin sensitization for

dimethylsulfoxide-treated cells incubated with toxin at

37C (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 lysoendo-somes, 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

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.

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 EC50 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 EC50values were calculated from four independent experiments with triplicate samples.

Toxin exposure

EC 50 (lgÆmL)1)

a

Cells treated with 25 lg saporinÆmL)1 exhibited approximately 85% of the protein synthesis levels recorded for unintoxicated con-trol cells.

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.

(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 4C

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 EC50

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

2

NH3

2Cl_

1

N

2

H2 N

NH3 + +

+

3Cl_

2

N

2

H2 N

N

2

NH3 +

+

4Cl_

3

Fig 5 Lipopolyamine structures The

molecular weights of 1–3, as previously

described [16], are 784, 891, and

999 gÆmol)1, respectively.

Cells coincubated with saporin and 5 lgÆmL)1of

lipo-polyamine were between 33- and 83-fold more sensitive

to saporin at the EC50value 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 concon-trols 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 intoxicatransfec-tion

Lipopolyamine treatment enhances the potency

of uPA-saporin, an anticancer toxin conjugate

Lipopolyamines are being developed as nontoxic

in vivogene 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 muuPA-sapo-rine 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)1of 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 efficiencya

Saporin sensitization

Ricin sensitization

Ricin A chain sensitization

a

Relative transfection efficiency as previously reported [16].bCells 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)1in the absence of lipopolyamine exhibited 22% of the protein synthesis levels recorded for unintoxicated control cells.

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 EC50 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

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.

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 aeruginosaexotoxin 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 37C and 5% CO2

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 37C and 5% CO2

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 [35S]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 [35S]methionineÆmL)1 for 15 min Radio-labeled cells were washed twice with ice-cold 10% trichloroacetic acid in NaCl⁄ Pi before solubilization in 0.2 N NaOH [35S]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

5 ngÆmL)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 EC50 These EC50values 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 EC50values Thus,

a report of ‘n-fold sensitivity’ represents the relative change