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Soapwort saponins trigger clathrin-mediated endocytosis of saporin, a type I ribosome-inactivating protein
Chemico-Biological Interactions 176 (2008) 204–211
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Soapwort saponins trigger clathrin-mediated endocytosis of saporin, a type I ribosome-inactivating protein A. Weng a , C. Bachran b , H. Fuchs b , M.F. Melzig a,∗ a b
Institute of Pharmacy, Free University Berlin, D-14195 Berlin, Germany Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Charité – Universitätsmedizin Berlin, Berlin, Germany
a r t i c l e
i n f o
Article history: Received 11 June 2008 Received in revised form 7 August 2008 Accepted 7 August 2008 Available online 15 August 2008 Keywords: Saporin Saponin Clathrin-mediated endocytosis
a b s t r a c t Saporin, a type I ribosome-inactivating protein (RIP), removes adenine residues from the 28S ribosomal RNA as part of a process that leads to inhibition of protein synthesis. However, as shown in this study, neither saporin nor his-tagged saporin (both 0.6–6 pM) exert toxicity on several human cell lines including H-2171, SK-N-SH, HEP-G2, MOLT-3, THP-1, HL-60 and ECV-304. Saporin and his-tagged saporin became highly cytotoxic when they were used in a combined treatment with Soapwort saponins (SA). When combined with SA (2–4 g/ml) saporin became as cytotoxic as the highly toxic type II RIP rViscumin reflected by an IC50 of 42.5 × 10−12 M for saporin and 21.5 × 10−12 M for rViscumin. We demonstrated that saporin was internalized via clathrin-mediated endocytosis, followed by the release into the endosomal transport system. Our results indicate that SA triggers this endocytic event rendering the otherwise cell membrane impermeable type I RIP saporin a potent cytotoxin. This effect was not cell line-specific suggesting that saporin exploits a common SA-dependent mechanism to enter cells. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Saponins in general are surface active compounds which give stable foams in water and were shown to effect the plasma membrane of living cells and model membranes by interacting with cholesterol. They are mainly produced by plants and of lower molecular weight (<2 kDa) [1]. The saponins which were used in this study are Soapwort saponins (SA) derived from Saponaria officinales L. SA have a five-ringed C30 backbone (aglycone) with two hydrophilic sugar units, attached at the 3 and 28 carbons of the hydrophobic aglycone [2]. As SA saporin is also present in S. officinales L. Saporin is a ribosome-inactivating protein (RIP) with 30 kDa, possessing RNA N-glycosidase activity. It removes the adenine residue at position 4324 from the 28S ribosomal RNA
∗ Corresponding author. Fax: +49 30 838 51461. E-mail address: [email protected] (M.F. Melzig). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.08.004
preventing protein synthesis [3]. Two types of RIPs exist characterized by either one subunit (type I RIPs) or two subunits (type II RIPs). In type II RIPs one subunit (B-chain) exhibits lectin activity and binds to terminal galactose residues on the cell surface and thereafter enables the catalytic subunit (A-chain) to enter cells. One of the most prominent type II RIPs is viscumin from Viscum album L. In contrast, type I RIPs only consists of the catalytic subunit (Achain), such as saporin or agrostin. They are considerably less cytotoxic due to the lack of the B-chain that mediates the cell membrane transfer of the A-chain in type II RIPs [4]. In previous studies we have shown that a combination of the cell membrane impermeable, ribosome-inactivating protein agrostin from Agrostemma githago L. with SA drastically enhanced the cytotoxicity of agrostin (>10,000-fold) and saporin (>100,000-fold) [2]. This was also shown for a type I-based immunotoxin [5]. However, the underlying mechanism of this synergistic cytotoxicity remained unclear.
A. Weng et al. / Chemico-Biological Interactions 176 (2008) 204–211
Since saponins were known as membrane active compounds, it was assumed that SA act mainly by interaction of the aglycone with the sterols (cholesterol) of the plasma membrane followed by the aggregation of the carbohydrate moieties. This may induce the rearrangement of the phospholipid bilayer and pore formation [6], possibly followed by translocation of type I RIPs through these pores. However, the cytotoxicity of agrostin/SA mixtures was hampered by latrunculin A [2], which inhibits actin polymerization and endocytosis [7]. It was therefore hypothesized that the increase in cytotoxicity of type I RIPs by SA was rather due to the induction of endocytosis than to an unspecific creation of SA-induced pores. Because nothing is known to date about the cell target specificity of saporin/SA mixtures, we investigated the cytotoxicity of unmodified saporin and histidinetagged saporin (His saporin) in combination with SA in several malignant cell lines of different origin: H-2171 (human small cell lung carcinoma), HEP-G2 (human hepatocellular carcinoma), SK-N-SH (human neuroblastoma), ECV-304 (human urinary bladder carcinoma), MOLT-3 (human T cell leukemia), THP-1 (human acute monocytic leukemia) and HL-60, a human acute myeloid leukemia cell line. Due to reports about protein impurities in RIPs from plants which possibly contribute to cytotoxicity [4] histidine-tagged saporin (His saporin) was especially used for labeling experiments with Alexa-Fluor 488. In order to illustrate the cytotoxicity-enhancing effect of SA on saporin, rViscumin, a highly toxic type II RIP, was used as highly efficient cytotoxic agent on human cells. To answer the question which mechanism allows the SA-induced penetration of unmodified saporin and His saporin into the cell, we scrutinized the effect of several inhibitors of endocytosis on the cytotoxicity of SA/saporin and SA/His saporin: chlorpromazine, imipramine and cyclosporine A (Cy-A) as inhibitors of clathrin-mediated endocytosis. Bafilomycin A1 was used as inhibitor of the endosomal transport and nocodazole as a microtubules disrupting agent. Filipin III and okadaic acid were used as inhibitors of caveollae-mediated endocytosis. The influence of these inhibitors on the internalization of Alexa-Fluor 488-labeled His saporin was also investigated.
2. Materials and methods Saporin, the tetrazolium salt 2,3-bis-(2-methoxy4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), phenazine methosulfate (PMS), bafilomycin A1, chlorpromazine, imipramine, cyclosporine A, nocodazole, filipin III and okadaic acid were purchased from Sigma (Steinheim, Germany). Alexa-Fluor 488 5-TFP was purchased from Invitrogen (Karlsruhe, Germany). BioGel P-30, fine was purchased from Bio-Rad (München, Germany). White saponin (Soapwort saponins), was purchased from Merck (Darmstadt, Germany). Recombinant Viscumin (rViscumin) was a generous gift from Cytavis BioPharma (Bergisch Gladbach, Germany).
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2.1. Cell culture HEP-G2 (DSMZ-No. ACC 180), MOLT-3 (DSMZ-No. ACC 84), THP-1 (DSMZ-No. ACC 16), H-2171 (DSMZ-No. ACC 16), HL-60 (DSMZ-No. ACC 3) and ECV-304 (DSMZ-No. ACC 304) cells were purchased from the German Cell Culture Collection (Braunschweig, Germany). SK-N-SH (ATCC-No. HTB-11) cells were purchased from the American Type Culture Collection (Manassas, USA). Except MOLT-3, H-2171, THP-1 and HL-60, all cell lines were cultured in modified Eagle’s medium (MEM) without phenol red supplemented with 15% fetal bovine serum (FBS). MOLT-3, HL-60 and THP1 cells were cultured in RPMI 1640 supplemented with 15% FBS, H-2171 cells in RPMI 1640 with 20% FBS. 2.2. Preparation of recombinant His saporin For protein expression plasmid DNA containing the cDNA of His saporin (in expression vector pET11d) was transformed into Escherichia coli strain Rosetta DE3 pLysS (Novagen, Schwalbach, Germany). Cloning of the protein is described elsewhere in detail [8]. Cells were grown overnight in Luria-Broth medium supplemented with 50 g/ml ampicillin at 37 ◦ C. 20 ml of the overnight culture were used to inoculate 400 ml Luria-Broth medium containing 50 g/ml ampicillin. After further growth at 37 ◦ C protein expression was induced at an optical density of A578 nm = 0.4–0.8 by adding 1 mM isopropyl-d-thiogalactopyranoside. After 3 h incubation cells were harvested by centrifugation (5000 × g, 4 ◦ C, 10 min), resuspended in phosphate-buffered saline (PBS; 150 mM sodium chloride, 8.33 mM disodium hydrogen phosphate, 1.67 mM potassium dihydrogen phosphate, pH 7.4) supplemented with 20 mM imidazole and stored at −20 ◦ C. Protein was released from bacterial cells by sonication on ice (5 × 20 pulses, duty cycle 4–7, sonifier 250, Branson, Danbury, USA) after thawing. Cell debris were removed by a further centrifugation (16,100 × g, 4 ◦ C, 30 min) and the supernatant applied to immobilized metal ion affinity chromatography by using a preequilibrated nickel–nitrilotriacetic acid–sepharose column (Qiagen, Hilden, Germany). Column-bound His saporin was washed with 5 ml PBS supplemented with 50 mM imidazole and 5 ml PBS with 70 mM imidazole. The protein was finally eluted by 5 ml PBS containing 250 mM imidazole. His saporin-containing fractions were pooled and 4× dialyzed against PBS overnight, thereafter concentrated by use of Amicon centrifugal filter devices (Millipore, Eschborn, Germany) with a molecular weight cut off of 10 kDa. Protein concentration was determined with the Advanced Protein Assay (Cytoskeleton, Denver, USA). Purity of His saporin was analyzed by applying 1 g of bovine serum albumin, 1 g wild type saporin and 1 g of His saporin to an SDS-PAGE with subsequent Coomassie staining. The enzymatic activity of His saporin was determined in a colorimetric adenine quantification assay [9]. The quality analysis of purified His saporin on a Coomassie-stained SDS-polyacrylamide gel revealed that the protein band appeared at the expected molecular weight (30 kDa) and the comparison with 1 g bovine serum albumin corroborates the determined protein con-
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centration (Fig. 2). The Coomassie staining furthermore demonstrates a purity of more than 90%. The enzymatic activity of the purified toxin was determined by a colorimetric adenine quantitation assay and found to be the same as of commercial untagged saporin (data not shown).
duced formazan was measured at 580 nm in a microplate reader (Tecan Spectra Fluor, Crailsheim, Germany). Each value of viability presented in the figures represents the mean of eight parallel experimental samples and refers to untreated controls.
2.3. Toxin labeling with Alexa-Fluor 488 5-TFP
2.6. Determination of IC50 of rViscumin and saporin/SA
50 l HCO3 − solution (pH 9) was added to 0.5 ml of a solution (2 mg/ml in PBS). 30 l Alexa-Fluor 5TFP (5 mg/ml in DMSO) solution was added and the mixture was swirled 1 h in the dark. The mixture was loaded to a BioGel P-30 column (0.32 × 7.8 in.) and purified by size exclusion chromatography with PBS as elution buffer. Eluted fractions were collected and protein concentration was determined by a bicinchoninic acid assay. Calibration was performed prior to the labeling procedure with unlabeled toxin. The labeled toxin remained fully active as determined by XTT assay.
2000 ECV-304 cells were seeded in 96-well plates. After 24 h the medium was aspirated and cells were washed with 100 l PBS per well. 83.5 l MEM without FBS was added and 10 l rViscumin solution (ranging from 1.7 × 10−10 M to 1.0 × 10−14 M) was added to each well. Dilutions of rViscumin were made in PBS containing 0.01% Tween 80. After 15 min 16.5 l FBS was added to each well. For the determination of the IC50 of saporin in combination with SA, ECV-304 cells were incubated with variable concentrations of saporin (ranging from 6.6 × 10−9 M to 4 × 10−13 M) and constant concentrations of SA (4 g/ml). All cells were further incubated for 72 h and viability was determined as described above.
His saporin
2.4. Fluorescence microscopy ECV-304 cells were seeded in Lab-Tek chamber slides (Nunc; New York, USA) at a density of approximately 4000 cells per chamber in 400 l MEM. After 24 h the medium was aspirated and cells were washed (400 l PBS). Thereafter 200 l MEM was added to each chamber. Cells were incubated with 20 l Alexa-Fluor 488-labeled His saporin (Alexa-His saporin) (6.5 × 10−10 M) either without SA or with 4 g/ml SA for 6 h. For experiments with inhibitors, ECV-304 cells were pre-incubated with 20 l chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M), okadaic acid (75 nM) or nocodazole (5 M) for 30 min. Afterwards medium was aspirated and cells were washed two times with 400 l PBS. Cells were examined using an epifluorescence microscope (BX 41, Olympus, Tokyo, Japan, 60× oil immersion). Images were acquired using a digital camera (DXM1200; Nikon, Tokyo, Japan) and the ACT-1 software. 2.5. Cell viability assay The cytotoxicity was determined in cell viability assays using the XTT reduction test with PMS as mediator. PMS is needed as an electron coupling reagent [10]. In brief, ECV-304, SK-N-SH and HEP-G2 cells were plated in 96-well plates at a density of 2000 cells per well in 100 l MEM with 15% FBS. The other cell lines (THP-1 7000 per well, MOLT-3 40,000 per well, H-2171 10,000 per well and HL-60 10,000 per well) were plated in 96-well plates in 100 l RPMI 1640 with 15% or 20% (H-2171 cells) FBS. All cells were cultivated in phenol red-free medium. A mixture of saporin (6 pM) and SA (4 g/ml) was added 24 h after plating to ECV-304, SKN-SH, H-2171, HL-60 and HEP-G2 cells. MOLT-3 and THP-1 cells were incubated with saporin (6 pM and 0.6 pM) and only 3 and 2 g/ml SA. Furthermore, the same experiments were performed with treatment of saporin without SA. After 72 h incubation, 50 l XTT solution (1 mg/ml) including 8 g/ml PMS was added to each well and cells were further incubated for 3 h at 37 ◦ C. Absorbance of the pro-
2.7. Cytotoxicity experiments with inhibitors of endocytosis 2000 ECV-304 cells per well were seeded in 96-well plates in 100 l MEM and grown 24 h. Cells were then pre-incubated with chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M) or okadaic acid (75 nM) for 30 min. After addition of a SA (4 g/ml)/saporin (6 pM) mixture, either with saporin or His saporin, cells were further incubated for 4 h. The medium was gently removed and cells were washed with 100 l PBS. Controls were only incubated with inhibitors for 4.5 h. 100 l MEM was added to each well and after a further incubation period (72 h) viability was determined with the XTT test as described above. 2.8. Statistics Data from the cytotoxic experiments were analyzed by Mann–Whitney test (U-test). 3. Results 3.1. Cell line-specific cytotoxicity of saporin/SA mixtures First, we investigated the cytotoxicity of saporin in combination with SA on different cell lines (Fig. 1). All cell lines were not affected by treatment with only saporin at the indicated concentrations (black columns). The appropriate mixtures of saporin with SA (white columns) were highly cytotoxic indicating an increased internalization of saporin into the cell. Exclusive SA incubation at the concentrations used for the combination with saporin showed no cytotoxicity in all cell lines (dotted columns). Since cytotoxicity of saporin/SA was observed in all cell lines, we assumed that saporin exploits the same mechanism in all cell lines to get into the cell. This mechanism was triggered by SA. Because until now cytotoxicity of type I RIPs with SA has been best characterized in ECV-304 cells [2,11] we used these cells
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Fig. 1. Cell line-specific cytotoxicity of saporin/SA mixtures. ECV-304, SK-N-SH, HEP-G2, H-2171, THP-1, MOLT-3 and HL-60 cells were plated in 96-well plates and 24 h thereafter 10 l of a saporin (6 pM)/SA (4 g/ml) mixture was added to ECV-304, SK-N-SH, H-2171, HL-60 and HEP-G2 cells. MOLT-3 cells were incubated with saporin (6 pM)/SA (3 g/ml) and THP-1 with saporin (0.6 pM)/SA (2 g/ml). After 72 h, viability was determined using the XTT reduction test. All cell lines were not affected by sole saporin treatment (black columns) and single SA at the indicated concentrations (dotted columns). The appropriate mixtures of saporin with SA (white columns) were highly cytotoxic. Each value represents the mean of eight experimental samples and refers to untreated control cells. *Significant to control (U-test: p ≤ 0.05).
exemplarily for further investigations on the SA-induced internalization of saporin into the cell. 3.2. IC50 of rViscumin and saporin with SA
hampered the cytotoxicity of the toxins in combination with SA indicating impaired transport of saporin within the endosomal system. Treatment with the inhibitors alone for the same incubation period did not result in reduced cell viabilities (Fig. 4).
In combination with SA (4 g/ml) the cytotoxicity of saporin was increased to a level similar to that of the type II RIP rViscumin. rViscumin achieved an IC50 value of 21.5 × 10−12 M on ECV-304 cells and the combination of saporin with SA 42.5 × 10−12 M (data not shown). 3.3. Decrease of cytotoxicity by different inhibitors of endocytosis ECV-304 cells were pre-incubated for 30 min with different inhibitors of endocytosis and treated thereafter with saporin (6 pM)/SA (4 g/ml) (Fig. 3, black columns) or His saporin (6 pM)/SA (4 g/ml) (Fig. 3, white columns). Cell viability was determined via the absorbance at 580 nm in the XTT assay. A toxicity index was defined as the absorbance ratio between cells pre-incubated with inhibitors before saporin/SA treatment and those directly exposed to saporin/SA (AInhibitor+S/SA /AS/SA ). Based on this definition the toxicity index represents the inhibitorinduced x-fold decrease of saporin/SA or His saporin/SA cytotoxicity. As depicted in Fig. 3 chlorpromazine, imipramine and cyclosporine A, all of it drugs known to affect clathrin-mediated endocytosis, significantly hampered the cytotoxicity of saporin and His saporin in combination with SA. In contrast, filipin III and okadaic acid, both known as inhibitors of caveolae-mediated endocytosis had no influence on the cytotoxicity of saporin/SA or His saporin/SA. Bafilomycin A1, a potent inhibitor of vacuolar ATPase, also
Fig. 2. Purity of His saporin. Coomassie-stained SDS-gel with 1 g His saporin, 1 g saporin and 1 g BSA. His Saporin appears at a molecular weight of 30 kDa. The result shows a small size difference between His saporin and saporin, due to the additional amino acids of the his-tag within His saporin.
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Fig. 3. Inhibition of saporin/SA cytotoxicity. ECV-304 cells were seeded in 96-well plates and grown for 24 h. Cells were then pre-incubated with chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M) or okadaic acid (75 nM) for 30 min. After addition of a SA (4 g/ml)/saporin (6 pM) or a His saporin (6 pM)/SA mixture, cells were incubated for 4 h. After washing (100 l PBS), 100 l MEM was added to each well and cells were further grown for 72 h. Viability was determined by XTT reduction test. The y-axis represents the x-fold decrease (referred to as toxicity index) of the appropriate cytotoxic mixtures caused by the additional pre-treatment with the appropriate inhibitor. *Significant to control (U-test: p ≤ 0.05).
3.4. Fluorescence microscopy with Alexa-Fluor 488-labeled His saporin To answer the question whether the cytotoxicity of saporin in combination with SA was associated with the enhanced translocation of the toxin through the cell membrane, we labeled His saporin with Alexa-Fluor 488 5-TFP. Due to reports about protein impurities in RIPs from plants, which possibly contribute to cytotoxicity [4], labeling was only performed for recombinant His saporin. ECV-304 cells were incubated with Alexa-Fluor 488labeled His saporin (Alexa-His saporin) (6.5 × 10−10 M) in combination with SA (4 g/ml). The green fluorescence of Alexa-His saporin could be detected within the cells (Fig. 5a). Cells treated only with Alexa-His saporin (6.5 × 10−10 M) without SA exhibited only weak green fluorescence (pic-
ture not shown). Based on this result we concluded that SA induced uptake and possibly endocytosis of His saporin. We next scrutinized the effect of several inhibitors on the endocytosis of Alexa-His saporin triggered by SA. Pre-incubation with chlorpromazine, imipramine and cyclosporine A hampered endocytosis of Alexa-His saporin nearly completely whereas filipin III and ocadaic acid had no inhibitory effect on the SA-induced internalization of Alexa-His saporin (picture not shown). The results obtained by fluorescence microscopy lead also to the conclusion that His saporin was internalized via clathrin-mediated endocytosis, since chlorpromazine, imipramine and cyclosporine A were shown to inhibit clathrin-mediated endocytosis (see discussion). We next analyzed the further transport of His saporin once the toxin was internalized. Cells were pre-incubated with bafilomycin A1 followed by the addition of AlexaHis saporin/SA. No fluorescence could be detected (picture not shown), indicating involvement of early and late endosomes in the transport of His saporin. This was confirmed by the results with the microtubules-disrupting agent nocodazole (Fig. 5b) that inhibits transport from early to late endosomes. As seen in Fig. 5b His saporin accumulated in larger vesicles after pre-incubation with nocodazole. 4. Discussion
Fig. 4. Cytotoxicity of the inhibitors of endocytosis. ECV-304 cells were seeded in 96-well plates and grown for 24 h. Cells were then incubated with chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M) or okadaic acid (75 nM) for 4.5 h. After washing (100 l PBS) 100 l MEM was added to each well and cells were further incubated for 72 h. Viability was determined by XTT reduction test. Each value represents the mean of eight experimental samples and refers to untreated control cells. *Significant to control (U-test: p ≤ 0.05).
In previous studies we have shown that cytotoxicity of type I ribosome-inactivating proteins like agrostin was strongly enhanced in ECV-304 cells after combination with special saponins (SA), derived from S. officinalis L. [2]. However, the mechanism of SA leading to the translocation of type I RIPs through the plasma membrane and the target cell specificity of the effect was unknown. Therefore we investigated the cytotoxicity of unmodified saporin and His saporin in combination with SA on several cell lines of different origin and performed experiments with several endocytosis-affecting inhibitors. As
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Fig. 5. Enhancement of toxin internalization by SA. ECV-304 cells were seeded in chamber slides and grown for 24 h. After washing, SA (4 g/ml) and Alexa-Fluor 488-labeled His saporin (Alexa-His saporin) (6.5 × 10−10 M) was added and cells were further incubated for 6 h (a). Cells were also incubated with nocodazole (5 M) for 30 min, followed by the addition of the SA (4 g/ml)/Alexa-His saporin (6.5 × 10−10 M) mixture. Cells were examined by an epifluorescence microscope, 60× oil immersion. Note the enhanced internalization of Alexa-His saporin (green fluorescence) in SA-treated cells (a). Pre-treatment with nocodazole (5 M) lead to an accumulation of His saporin in larger vesicles (b). The bar in (b) represents the scale the fluorescence photographs.
shown in Fig. 2, saporin became highly cytotoxic when it was used in a combined treatment with SA on all tested cell lines. As a result we assumed that saporin exploits the same SA-mediated mechanism in all mammalian cells to translocate through the plasma membrane. This implicates that saporin binds either to a membrane receptor expressed by all cell lines investigated here or to serum components that gain entrance into the cell via endocytosis as component-saporin-complexes. Binding to a soluble component like ␣-macroglobulin could result in in situ formation of a complex that may enter cells via ␣-macroglobulin receptor. Such complex formation was shown for the ricin A chain that forms high molecular complexes with ␣-macroglobulin under cell culture conditions and in vivo [12]. However in previous studies it was shown that cytotoxicity of
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saporin is not mediated through ␣-macroglobulin receptor [13]. To illustrate the potentiation of cytotoxicity of saporin by SA we compared the cytotoxicity of saporin in combination with SA to the cytotoxicity of rViscumin, a highly toxic type II RIP from V. album L. [14]. When combined with SA, saporin became as cytotoxic as the type II RIP rViscumin reflected by an IC50 of 42.5 × 10−12 M for saporin/SA and 21.5 × 10−12 M for rViscumin. Thus, SA delivers saporin as effective into the cell as the B chain of the type II RIP rViscumin. However, there is no structural homology between the B-chain of rViscumin which binds via amino acids to the cell [15] and SA, indicating different mechanisms of toxin entry into the cell. We investigated the SA-triggered internalization of saporin in detail with several endocytosis inhibitors, which are known to affect the clathrin- and caveolae-mediated endocytosis. Filipin III from Streptomyces filipinensis and okadaic acid from Prorocentrum concavum, both known as inhibitors of caveolae-mediated endocytosis [16–18] had no influence on toxicity of unmodified and His saporin (Fig. 3), both in combination with SA. In contrast, chlorpromazine and imipramine, both known as inhibitors of clathrin-mediated endocytosis, hampered cytotoxicity of saporin and His saporin with SA (Fig. 3) significantly. In addition endocytosis of Alexa-His saporin was also markedly reduced when cells were pre-treated with chlorpromazine or imipramine (panel not shown). Chlorpromazine and imipramine cause considerable decrease of clathrin-coated pits at the cell surface, paralleled by redistribution of the adaptor protein 2 (AP-2) into the cytoplasm [19]. AP-2 is required to keep the clathrin lattice at the plasma membrane. In addition, the internalization of several viruses known to be endocytosed by clathrin-mediated endocytosis was hampered upon chlorpromazine (50–100 M) and imipramine treatment [20,21]. The phosphatase calcineurin (Cn) was shown to dephosphorylate endocytic proteins of the clathrin-mediated endocytosis machinery like amphiphysin I/II, synaptojanin and epsin [22]. It was further shown that Cn binds to dynamin and that disruption of Cn and dynamin leads to the inhibition of clathrin-mediated endocytosis [23]. Cyclosporine A, a non-polar cyclic oligopeptide produced by the fungus Tolypocladium inflatum was shown to inactivate Cn [24,25] followed by inhibition of endocytosis [26,27]. As depicted in Fig. 3, cytotoxicity of saporin/SA and His saporin/SA was hampered by Cy-A. SA-mediated internalization of Alexa-His saporin was also impeded upon Cy-A treatment (panel not shown). We regarded this as a further proof for the role of SA on clathrin-mediated endocytosis of saporin. Having successfully entered a cell saporin has to escape from the endosome in order to reach its target, the ribosomes in the cytosol. To scrutinize which parts of the endosomal system are involved in the transport of saporin we investigated the effect of bafilomycin A1, a macrolid antibiotic, on the cytotoxicity of saporin in combination with SA. Bafilomycin A1 specifically inhibits the vacuolar ATPase [28] leading to an increase of the endosomal and lysosomal pH. Endosomal acidification is necessary for regular endosomal transport [29,30] and for receptor
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recycling [31,32]. As illustrated in Fig. 3, pre-incubation with bafilomycin A1 inhibited the enhancing effect of SA on saporin cytotoxicity. Internalization of Alexa-His saporin was almost completely inhibited after bafilomycin A1 treatment (panel not shown). This effect was either due to inhibition of receptor-mediated endocytosis as shown for bafilomycin A1-treated cells [33] or due to a transport block from early to late endosomes, followed by a reduced release of saporin molecules into the cytosol. Especially the accumulation of Alexa-His saporin in larger vesicles after incubation with the microtubule-disrupting agent nocodazole (Fig. 5b) strongly supports this assumption since nocodazole blocked transport from early to late endosomes [29,34–36]. Thus, we expect saporin to escape from the late endosomes into the cytosol and since the inhibitors prevented saporin from arriving at the late endosomes the resulting cytotoxicity is lower. We demonstrated in this study that synergistic cytotoxicity between saporin and SA is based on the induction of endocytosis of saporin, triggered by SA. A direct chemical interaction between SA and saporin is unlikely since preincubation with SA without saporin for 1 h, followed by an extensive wash out of SA lead not to a decrease of cytotoxicity of saporin (data not shown). We further observed that the effect of SA on saporin toxicity could not be enhanced by higher concentrations of SA. It followed an “all or none” principle. Therefore it seems to be a kind of SA-induced transient change of the membrane properties which lead to a sensitization against saporin. It was further evidenced that internalization of saporin most likely occurs via clathrin-mediated endocytosis, followed by the release of saporin into the endosomal transport system and toxin release into the cytosol. Synergistic cytotoxicity between saporin and SA is therefore not a consequence of the generally postulated surface activity of saponins, leading to the formation of pores in the cell membrane [6,37]. However, it is conceivable that SA play a role as a kind of anchor by association with membrane components like cholesterol. This would lead to the insertion of SA molecules into the plasma membrane. The remaining sugar tails could interact with specific endocytic factors, followed by the induction of endocytosis of receptor bound saporin. Enhancement of cytotoxicity of saporin by SA was shown in several malignant cell types formerly derived from the lung (H-1217), brain (SK-N-SH), bladder (ECV304), primary immune system (THP-1, HL-60) and secondary immune system (MOLT-3). Since we have shown that SA trigger clathrin-mediated endocytosis of saporin, we assumed that this would also toke place in normal cells. An increased saporin internalization would lead to strong cytotoxicity in normal cells which limits the use of saporin/SA for therapeutic use. Conflict of interest None. Acknowledgement Funding: German Research Foundation (FU 408/3-1).
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Soapwort saponins trigger clathrin-mediated endocytosis of saporin, a type I ribosome-inactivating protein A. Weng a , C. Bachran b , H. Fuchs b , M.F. Melzig a,∗ a b
Institute of Pharmacy, Free University Berlin, D-14195 Berlin, Germany Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Charité – Universitätsmedizin Berlin, Berlin, Germany
a r t i c l e
i n f o
Article history: Received 11 June 2008 Received in revised form 7 August 2008 Accepted 7 August 2008 Available online 15 August 2008 Keywords: Saporin Saponin Clathrin-mediated endocytosis
a b s t r a c t Saporin, a type I ribosome-inactivating protein (RIP), removes adenine residues from the 28S ribosomal RNA as part of a process that leads to inhibition of protein synthesis. However, as shown in this study, neither saporin nor his-tagged saporin (both 0.6–6 pM) exert toxicity on several human cell lines including H-2171, SK-N-SH, HEP-G2, MOLT-3, THP-1, HL-60 and ECV-304. Saporin and his-tagged saporin became highly cytotoxic when they were used in a combined treatment with Soapwort saponins (SA). When combined with SA (2–4 g/ml) saporin became as cytotoxic as the highly toxic type II RIP rViscumin reflected by an IC50 of 42.5 × 10−12 M for saporin and 21.5 × 10−12 M for rViscumin. We demonstrated that saporin was internalized via clathrin-mediated endocytosis, followed by the release into the endosomal transport system. Our results indicate that SA triggers this endocytic event rendering the otherwise cell membrane impermeable type I RIP saporin a potent cytotoxin. This effect was not cell line-specific suggesting that saporin exploits a common SA-dependent mechanism to enter cells. © 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Saponins in general are surface active compounds which give stable foams in water and were shown to effect the plasma membrane of living cells and model membranes by interacting with cholesterol. They are mainly produced by plants and of lower molecular weight (<2 kDa) [1]. The saponins which were used in this study are Soapwort saponins (SA) derived from Saponaria officinales L. SA have a five-ringed C30 backbone (aglycone) with two hydrophilic sugar units, attached at the 3 and 28 carbons of the hydrophobic aglycone [2]. As SA saporin is also present in S. officinales L. Saporin is a ribosome-inactivating protein (RIP) with 30 kDa, possessing RNA N-glycosidase activity. It removes the adenine residue at position 4324 from the 28S ribosomal RNA
∗ Corresponding author. Fax: +49 30 838 51461. E-mail address: [email protected] (M.F. Melzig). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.08.004
preventing protein synthesis [3]. Two types of RIPs exist characterized by either one subunit (type I RIPs) or two subunits (type II RIPs). In type II RIPs one subunit (B-chain) exhibits lectin activity and binds to terminal galactose residues on the cell surface and thereafter enables the catalytic subunit (A-chain) to enter cells. One of the most prominent type II RIPs is viscumin from Viscum album L. In contrast, type I RIPs only consists of the catalytic subunit (Achain), such as saporin or agrostin. They are considerably less cytotoxic due to the lack of the B-chain that mediates the cell membrane transfer of the A-chain in type II RIPs [4]. In previous studies we have shown that a combination of the cell membrane impermeable, ribosome-inactivating protein agrostin from Agrostemma githago L. with SA drastically enhanced the cytotoxicity of agrostin (>10,000-fold) and saporin (>100,000-fold) [2]. This was also shown for a type I-based immunotoxin [5]. However, the underlying mechanism of this synergistic cytotoxicity remained unclear.
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Since saponins were known as membrane active compounds, it was assumed that SA act mainly by interaction of the aglycone with the sterols (cholesterol) of the plasma membrane followed by the aggregation of the carbohydrate moieties. This may induce the rearrangement of the phospholipid bilayer and pore formation [6], possibly followed by translocation of type I RIPs through these pores. However, the cytotoxicity of agrostin/SA mixtures was hampered by latrunculin A [2], which inhibits actin polymerization and endocytosis [7]. It was therefore hypothesized that the increase in cytotoxicity of type I RIPs by SA was rather due to the induction of endocytosis than to an unspecific creation of SA-induced pores. Because nothing is known to date about the cell target specificity of saporin/SA mixtures, we investigated the cytotoxicity of unmodified saporin and histidinetagged saporin (His saporin) in combination with SA in several malignant cell lines of different origin: H-2171 (human small cell lung carcinoma), HEP-G2 (human hepatocellular carcinoma), SK-N-SH (human neuroblastoma), ECV-304 (human urinary bladder carcinoma), MOLT-3 (human T cell leukemia), THP-1 (human acute monocytic leukemia) and HL-60, a human acute myeloid leukemia cell line. Due to reports about protein impurities in RIPs from plants which possibly contribute to cytotoxicity [4] histidine-tagged saporin (His saporin) was especially used for labeling experiments with Alexa-Fluor 488. In order to illustrate the cytotoxicity-enhancing effect of SA on saporin, rViscumin, a highly toxic type II RIP, was used as highly efficient cytotoxic agent on human cells. To answer the question which mechanism allows the SA-induced penetration of unmodified saporin and His saporin into the cell, we scrutinized the effect of several inhibitors of endocytosis on the cytotoxicity of SA/saporin and SA/His saporin: chlorpromazine, imipramine and cyclosporine A (Cy-A) as inhibitors of clathrin-mediated endocytosis. Bafilomycin A1 was used as inhibitor of the endosomal transport and nocodazole as a microtubules disrupting agent. Filipin III and okadaic acid were used as inhibitors of caveollae-mediated endocytosis. The influence of these inhibitors on the internalization of Alexa-Fluor 488-labeled His saporin was also investigated.
2. Materials and methods Saporin, the tetrazolium salt 2,3-bis-(2-methoxy4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), phenazine methosulfate (PMS), bafilomycin A1, chlorpromazine, imipramine, cyclosporine A, nocodazole, filipin III and okadaic acid were purchased from Sigma (Steinheim, Germany). Alexa-Fluor 488 5-TFP was purchased from Invitrogen (Karlsruhe, Germany). BioGel P-30, fine was purchased from Bio-Rad (München, Germany). White saponin (Soapwort saponins), was purchased from Merck (Darmstadt, Germany). Recombinant Viscumin (rViscumin) was a generous gift from Cytavis BioPharma (Bergisch Gladbach, Germany).
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2.1. Cell culture HEP-G2 (DSMZ-No. ACC 180), MOLT-3 (DSMZ-No. ACC 84), THP-1 (DSMZ-No. ACC 16), H-2171 (DSMZ-No. ACC 16), HL-60 (DSMZ-No. ACC 3) and ECV-304 (DSMZ-No. ACC 304) cells were purchased from the German Cell Culture Collection (Braunschweig, Germany). SK-N-SH (ATCC-No. HTB-11) cells were purchased from the American Type Culture Collection (Manassas, USA). Except MOLT-3, H-2171, THP-1 and HL-60, all cell lines were cultured in modified Eagle’s medium (MEM) without phenol red supplemented with 15% fetal bovine serum (FBS). MOLT-3, HL-60 and THP1 cells were cultured in RPMI 1640 supplemented with 15% FBS, H-2171 cells in RPMI 1640 with 20% FBS. 2.2. Preparation of recombinant His saporin For protein expression plasmid DNA containing the cDNA of His saporin (in expression vector pET11d) was transformed into Escherichia coli strain Rosetta DE3 pLysS (Novagen, Schwalbach, Germany). Cloning of the protein is described elsewhere in detail [8]. Cells were grown overnight in Luria-Broth medium supplemented with 50 g/ml ampicillin at 37 ◦ C. 20 ml of the overnight culture were used to inoculate 400 ml Luria-Broth medium containing 50 g/ml ampicillin. After further growth at 37 ◦ C protein expression was induced at an optical density of A578 nm = 0.4–0.8 by adding 1 mM isopropyl-d-thiogalactopyranoside. After 3 h incubation cells were harvested by centrifugation (5000 × g, 4 ◦ C, 10 min), resuspended in phosphate-buffered saline (PBS; 150 mM sodium chloride, 8.33 mM disodium hydrogen phosphate, 1.67 mM potassium dihydrogen phosphate, pH 7.4) supplemented with 20 mM imidazole and stored at −20 ◦ C. Protein was released from bacterial cells by sonication on ice (5 × 20 pulses, duty cycle 4–7, sonifier 250, Branson, Danbury, USA) after thawing. Cell debris were removed by a further centrifugation (16,100 × g, 4 ◦ C, 30 min) and the supernatant applied to immobilized metal ion affinity chromatography by using a preequilibrated nickel–nitrilotriacetic acid–sepharose column (Qiagen, Hilden, Germany). Column-bound His saporin was washed with 5 ml PBS supplemented with 50 mM imidazole and 5 ml PBS with 70 mM imidazole. The protein was finally eluted by 5 ml PBS containing 250 mM imidazole. His saporin-containing fractions were pooled and 4× dialyzed against PBS overnight, thereafter concentrated by use of Amicon centrifugal filter devices (Millipore, Eschborn, Germany) with a molecular weight cut off of 10 kDa. Protein concentration was determined with the Advanced Protein Assay (Cytoskeleton, Denver, USA). Purity of His saporin was analyzed by applying 1 g of bovine serum albumin, 1 g wild type saporin and 1 g of His saporin to an SDS-PAGE with subsequent Coomassie staining. The enzymatic activity of His saporin was determined in a colorimetric adenine quantification assay [9]. The quality analysis of purified His saporin on a Coomassie-stained SDS-polyacrylamide gel revealed that the protein band appeared at the expected molecular weight (30 kDa) and the comparison with 1 g bovine serum albumin corroborates the determined protein con-
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centration (Fig. 2). The Coomassie staining furthermore demonstrates a purity of more than 90%. The enzymatic activity of the purified toxin was determined by a colorimetric adenine quantitation assay and found to be the same as of commercial untagged saporin (data not shown).
duced formazan was measured at 580 nm in a microplate reader (Tecan Spectra Fluor, Crailsheim, Germany). Each value of viability presented in the figures represents the mean of eight parallel experimental samples and refers to untreated controls.
2.3. Toxin labeling with Alexa-Fluor 488 5-TFP
2.6. Determination of IC50 of rViscumin and saporin/SA
50 l HCO3 − solution (pH 9) was added to 0.5 ml of a solution (2 mg/ml in PBS). 30 l Alexa-Fluor 5TFP (5 mg/ml in DMSO) solution was added and the mixture was swirled 1 h in the dark. The mixture was loaded to a BioGel P-30 column (0.32 × 7.8 in.) and purified by size exclusion chromatography with PBS as elution buffer. Eluted fractions were collected and protein concentration was determined by a bicinchoninic acid assay. Calibration was performed prior to the labeling procedure with unlabeled toxin. The labeled toxin remained fully active as determined by XTT assay.
2000 ECV-304 cells were seeded in 96-well plates. After 24 h the medium was aspirated and cells were washed with 100 l PBS per well. 83.5 l MEM without FBS was added and 10 l rViscumin solution (ranging from 1.7 × 10−10 M to 1.0 × 10−14 M) was added to each well. Dilutions of rViscumin were made in PBS containing 0.01% Tween 80. After 15 min 16.5 l FBS was added to each well. For the determination of the IC50 of saporin in combination with SA, ECV-304 cells were incubated with variable concentrations of saporin (ranging from 6.6 × 10−9 M to 4 × 10−13 M) and constant concentrations of SA (4 g/ml). All cells were further incubated for 72 h and viability was determined as described above.
His saporin
2.4. Fluorescence microscopy ECV-304 cells were seeded in Lab-Tek chamber slides (Nunc; New York, USA) at a density of approximately 4000 cells per chamber in 400 l MEM. After 24 h the medium was aspirated and cells were washed (400 l PBS). Thereafter 200 l MEM was added to each chamber. Cells were incubated with 20 l Alexa-Fluor 488-labeled His saporin (Alexa-His saporin) (6.5 × 10−10 M) either without SA or with 4 g/ml SA for 6 h. For experiments with inhibitors, ECV-304 cells were pre-incubated with 20 l chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M), okadaic acid (75 nM) or nocodazole (5 M) for 30 min. Afterwards medium was aspirated and cells were washed two times with 400 l PBS. Cells were examined using an epifluorescence microscope (BX 41, Olympus, Tokyo, Japan, 60× oil immersion). Images were acquired using a digital camera (DXM1200; Nikon, Tokyo, Japan) and the ACT-1 software. 2.5. Cell viability assay The cytotoxicity was determined in cell viability assays using the XTT reduction test with PMS as mediator. PMS is needed as an electron coupling reagent [10]. In brief, ECV-304, SK-N-SH and HEP-G2 cells were plated in 96-well plates at a density of 2000 cells per well in 100 l MEM with 15% FBS. The other cell lines (THP-1 7000 per well, MOLT-3 40,000 per well, H-2171 10,000 per well and HL-60 10,000 per well) were plated in 96-well plates in 100 l RPMI 1640 with 15% or 20% (H-2171 cells) FBS. All cells were cultivated in phenol red-free medium. A mixture of saporin (6 pM) and SA (4 g/ml) was added 24 h after plating to ECV-304, SKN-SH, H-2171, HL-60 and HEP-G2 cells. MOLT-3 and THP-1 cells were incubated with saporin (6 pM and 0.6 pM) and only 3 and 2 g/ml SA. Furthermore, the same experiments were performed with treatment of saporin without SA. After 72 h incubation, 50 l XTT solution (1 mg/ml) including 8 g/ml PMS was added to each well and cells were further incubated for 3 h at 37 ◦ C. Absorbance of the pro-
2.7. Cytotoxicity experiments with inhibitors of endocytosis 2000 ECV-304 cells per well were seeded in 96-well plates in 100 l MEM and grown 24 h. Cells were then pre-incubated with chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M) or okadaic acid (75 nM) for 30 min. After addition of a SA (4 g/ml)/saporin (6 pM) mixture, either with saporin or His saporin, cells were further incubated for 4 h. The medium was gently removed and cells were washed with 100 l PBS. Controls were only incubated with inhibitors for 4.5 h. 100 l MEM was added to each well and after a further incubation period (72 h) viability was determined with the XTT test as described above. 2.8. Statistics Data from the cytotoxic experiments were analyzed by Mann–Whitney test (U-test). 3. Results 3.1. Cell line-specific cytotoxicity of saporin/SA mixtures First, we investigated the cytotoxicity of saporin in combination with SA on different cell lines (Fig. 1). All cell lines were not affected by treatment with only saporin at the indicated concentrations (black columns). The appropriate mixtures of saporin with SA (white columns) were highly cytotoxic indicating an increased internalization of saporin into the cell. Exclusive SA incubation at the concentrations used for the combination with saporin showed no cytotoxicity in all cell lines (dotted columns). Since cytotoxicity of saporin/SA was observed in all cell lines, we assumed that saporin exploits the same mechanism in all cell lines to get into the cell. This mechanism was triggered by SA. Because until now cytotoxicity of type I RIPs with SA has been best characterized in ECV-304 cells [2,11] we used these cells
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Fig. 1. Cell line-specific cytotoxicity of saporin/SA mixtures. ECV-304, SK-N-SH, HEP-G2, H-2171, THP-1, MOLT-3 and HL-60 cells were plated in 96-well plates and 24 h thereafter 10 l of a saporin (6 pM)/SA (4 g/ml) mixture was added to ECV-304, SK-N-SH, H-2171, HL-60 and HEP-G2 cells. MOLT-3 cells were incubated with saporin (6 pM)/SA (3 g/ml) and THP-1 with saporin (0.6 pM)/SA (2 g/ml). After 72 h, viability was determined using the XTT reduction test. All cell lines were not affected by sole saporin treatment (black columns) and single SA at the indicated concentrations (dotted columns). The appropriate mixtures of saporin with SA (white columns) were highly cytotoxic. Each value represents the mean of eight experimental samples and refers to untreated control cells. *Significant to control (U-test: p ≤ 0.05).
exemplarily for further investigations on the SA-induced internalization of saporin into the cell. 3.2. IC50 of rViscumin and saporin with SA
hampered the cytotoxicity of the toxins in combination with SA indicating impaired transport of saporin within the endosomal system. Treatment with the inhibitors alone for the same incubation period did not result in reduced cell viabilities (Fig. 4).
In combination with SA (4 g/ml) the cytotoxicity of saporin was increased to a level similar to that of the type II RIP rViscumin. rViscumin achieved an IC50 value of 21.5 × 10−12 M on ECV-304 cells and the combination of saporin with SA 42.5 × 10−12 M (data not shown). 3.3. Decrease of cytotoxicity by different inhibitors of endocytosis ECV-304 cells were pre-incubated for 30 min with different inhibitors of endocytosis and treated thereafter with saporin (6 pM)/SA (4 g/ml) (Fig. 3, black columns) or His saporin (6 pM)/SA (4 g/ml) (Fig. 3, white columns). Cell viability was determined via the absorbance at 580 nm in the XTT assay. A toxicity index was defined as the absorbance ratio between cells pre-incubated with inhibitors before saporin/SA treatment and those directly exposed to saporin/SA (AInhibitor+S/SA /AS/SA ). Based on this definition the toxicity index represents the inhibitorinduced x-fold decrease of saporin/SA or His saporin/SA cytotoxicity. As depicted in Fig. 3 chlorpromazine, imipramine and cyclosporine A, all of it drugs known to affect clathrin-mediated endocytosis, significantly hampered the cytotoxicity of saporin and His saporin in combination with SA. In contrast, filipin III and okadaic acid, both known as inhibitors of caveolae-mediated endocytosis had no influence on the cytotoxicity of saporin/SA or His saporin/SA. Bafilomycin A1, a potent inhibitor of vacuolar ATPase, also
Fig. 2. Purity of His saporin. Coomassie-stained SDS-gel with 1 g His saporin, 1 g saporin and 1 g BSA. His Saporin appears at a molecular weight of 30 kDa. The result shows a small size difference between His saporin and saporin, due to the additional amino acids of the his-tag within His saporin.
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Fig. 3. Inhibition of saporin/SA cytotoxicity. ECV-304 cells were seeded in 96-well plates and grown for 24 h. Cells were then pre-incubated with chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M) or okadaic acid (75 nM) for 30 min. After addition of a SA (4 g/ml)/saporin (6 pM) or a His saporin (6 pM)/SA mixture, cells were incubated for 4 h. After washing (100 l PBS), 100 l MEM was added to each well and cells were further grown for 72 h. Viability was determined by XTT reduction test. The y-axis represents the x-fold decrease (referred to as toxicity index) of the appropriate cytotoxic mixtures caused by the additional pre-treatment with the appropriate inhibitor. *Significant to control (U-test: p ≤ 0.05).
3.4. Fluorescence microscopy with Alexa-Fluor 488-labeled His saporin To answer the question whether the cytotoxicity of saporin in combination with SA was associated with the enhanced translocation of the toxin through the cell membrane, we labeled His saporin with Alexa-Fluor 488 5-TFP. Due to reports about protein impurities in RIPs from plants, which possibly contribute to cytotoxicity [4], labeling was only performed for recombinant His saporin. ECV-304 cells were incubated with Alexa-Fluor 488labeled His saporin (Alexa-His saporin) (6.5 × 10−10 M) in combination with SA (4 g/ml). The green fluorescence of Alexa-His saporin could be detected within the cells (Fig. 5a). Cells treated only with Alexa-His saporin (6.5 × 10−10 M) without SA exhibited only weak green fluorescence (pic-
ture not shown). Based on this result we concluded that SA induced uptake and possibly endocytosis of His saporin. We next scrutinized the effect of several inhibitors on the endocytosis of Alexa-His saporin triggered by SA. Pre-incubation with chlorpromazine, imipramine and cyclosporine A hampered endocytosis of Alexa-His saporin nearly completely whereas filipin III and ocadaic acid had no inhibitory effect on the SA-induced internalization of Alexa-His saporin (picture not shown). The results obtained by fluorescence microscopy lead also to the conclusion that His saporin was internalized via clathrin-mediated endocytosis, since chlorpromazine, imipramine and cyclosporine A were shown to inhibit clathrin-mediated endocytosis (see discussion). We next analyzed the further transport of His saporin once the toxin was internalized. Cells were pre-incubated with bafilomycin A1 followed by the addition of AlexaHis saporin/SA. No fluorescence could be detected (picture not shown), indicating involvement of early and late endosomes in the transport of His saporin. This was confirmed by the results with the microtubules-disrupting agent nocodazole (Fig. 5b) that inhibits transport from early to late endosomes. As seen in Fig. 5b His saporin accumulated in larger vesicles after pre-incubation with nocodazole. 4. Discussion
Fig. 4. Cytotoxicity of the inhibitors of endocytosis. ECV-304 cells were seeded in 96-well plates and grown for 24 h. Cells were then incubated with chlorpromazine (60 M), imipramine (60 M), bafilomycin A1 (20 nM), cyclosporine A (2.5 M), filipin III (3 M) or okadaic acid (75 nM) for 4.5 h. After washing (100 l PBS) 100 l MEM was added to each well and cells were further incubated for 72 h. Viability was determined by XTT reduction test. Each value represents the mean of eight experimental samples and refers to untreated control cells. *Significant to control (U-test: p ≤ 0.05).
In previous studies we have shown that cytotoxicity of type I ribosome-inactivating proteins like agrostin was strongly enhanced in ECV-304 cells after combination with special saponins (SA), derived from S. officinalis L. [2]. However, the mechanism of SA leading to the translocation of type I RIPs through the plasma membrane and the target cell specificity of the effect was unknown. Therefore we investigated the cytotoxicity of unmodified saporin and His saporin in combination with SA on several cell lines of different origin and performed experiments with several endocytosis-affecting inhibitors. As
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Fig. 5. Enhancement of toxin internalization by SA. ECV-304 cells were seeded in chamber slides and grown for 24 h. After washing, SA (4 g/ml) and Alexa-Fluor 488-labeled His saporin (Alexa-His saporin) (6.5 × 10−10 M) was added and cells were further incubated for 6 h (a). Cells were also incubated with nocodazole (5 M) for 30 min, followed by the addition of the SA (4 g/ml)/Alexa-His saporin (6.5 × 10−10 M) mixture. Cells were examined by an epifluorescence microscope, 60× oil immersion. Note the enhanced internalization of Alexa-His saporin (green fluorescence) in SA-treated cells (a). Pre-treatment with nocodazole (5 M) lead to an accumulation of His saporin in larger vesicles (b). The bar in (b) represents the scale the fluorescence photographs.
shown in Fig. 2, saporin became highly cytotoxic when it was used in a combined treatment with SA on all tested cell lines. As a result we assumed that saporin exploits the same SA-mediated mechanism in all mammalian cells to translocate through the plasma membrane. This implicates that saporin binds either to a membrane receptor expressed by all cell lines investigated here or to serum components that gain entrance into the cell via endocytosis as component-saporin-complexes. Binding to a soluble component like ␣-macroglobulin could result in in situ formation of a complex that may enter cells via ␣-macroglobulin receptor. Such complex formation was shown for the ricin A chain that forms high molecular complexes with ␣-macroglobulin under cell culture conditions and in vivo [12]. However in previous studies it was shown that cytotoxicity of
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saporin is not mediated through ␣-macroglobulin receptor [13]. To illustrate the potentiation of cytotoxicity of saporin by SA we compared the cytotoxicity of saporin in combination with SA to the cytotoxicity of rViscumin, a highly toxic type II RIP from V. album L. [14]. When combined with SA, saporin became as cytotoxic as the type II RIP rViscumin reflected by an IC50 of 42.5 × 10−12 M for saporin/SA and 21.5 × 10−12 M for rViscumin. Thus, SA delivers saporin as effective into the cell as the B chain of the type II RIP rViscumin. However, there is no structural homology between the B-chain of rViscumin which binds via amino acids to the cell [15] and SA, indicating different mechanisms of toxin entry into the cell. We investigated the SA-triggered internalization of saporin in detail with several endocytosis inhibitors, which are known to affect the clathrin- and caveolae-mediated endocytosis. Filipin III from Streptomyces filipinensis and okadaic acid from Prorocentrum concavum, both known as inhibitors of caveolae-mediated endocytosis [16–18] had no influence on toxicity of unmodified and His saporin (Fig. 3), both in combination with SA. In contrast, chlorpromazine and imipramine, both known as inhibitors of clathrin-mediated endocytosis, hampered cytotoxicity of saporin and His saporin with SA (Fig. 3) significantly. In addition endocytosis of Alexa-His saporin was also markedly reduced when cells were pre-treated with chlorpromazine or imipramine (panel not shown). Chlorpromazine and imipramine cause considerable decrease of clathrin-coated pits at the cell surface, paralleled by redistribution of the adaptor protein 2 (AP-2) into the cytoplasm [19]. AP-2 is required to keep the clathrin lattice at the plasma membrane. In addition, the internalization of several viruses known to be endocytosed by clathrin-mediated endocytosis was hampered upon chlorpromazine (50–100 M) and imipramine treatment [20,21]. The phosphatase calcineurin (Cn) was shown to dephosphorylate endocytic proteins of the clathrin-mediated endocytosis machinery like amphiphysin I/II, synaptojanin and epsin [22]. It was further shown that Cn binds to dynamin and that disruption of Cn and dynamin leads to the inhibition of clathrin-mediated endocytosis [23]. Cyclosporine A, a non-polar cyclic oligopeptide produced by the fungus Tolypocladium inflatum was shown to inactivate Cn [24,25] followed by inhibition of endocytosis [26,27]. As depicted in Fig. 3, cytotoxicity of saporin/SA and His saporin/SA was hampered by Cy-A. SA-mediated internalization of Alexa-His saporin was also impeded upon Cy-A treatment (panel not shown). We regarded this as a further proof for the role of SA on clathrin-mediated endocytosis of saporin. Having successfully entered a cell saporin has to escape from the endosome in order to reach its target, the ribosomes in the cytosol. To scrutinize which parts of the endosomal system are involved in the transport of saporin we investigated the effect of bafilomycin A1, a macrolid antibiotic, on the cytotoxicity of saporin in combination with SA. Bafilomycin A1 specifically inhibits the vacuolar ATPase [28] leading to an increase of the endosomal and lysosomal pH. Endosomal acidification is necessary for regular endosomal transport [29,30] and for receptor
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recycling [31,32]. As illustrated in Fig. 3, pre-incubation with bafilomycin A1 inhibited the enhancing effect of SA on saporin cytotoxicity. Internalization of Alexa-His saporin was almost completely inhibited after bafilomycin A1 treatment (panel not shown). This effect was either due to inhibition of receptor-mediated endocytosis as shown for bafilomycin A1-treated cells [33] or due to a transport block from early to late endosomes, followed by a reduced release of saporin molecules into the cytosol. Especially the accumulation of Alexa-His saporin in larger vesicles after incubation with the microtubule-disrupting agent nocodazole (Fig. 5b) strongly supports this assumption since nocodazole blocked transport from early to late endosomes [29,34–36]. Thus, we expect saporin to escape from the late endosomes into the cytosol and since the inhibitors prevented saporin from arriving at the late endosomes the resulting cytotoxicity is lower. We demonstrated in this study that synergistic cytotoxicity between saporin and SA is based on the induction of endocytosis of saporin, triggered by SA. A direct chemical interaction between SA and saporin is unlikely since preincubation with SA without saporin for 1 h, followed by an extensive wash out of SA lead not to a decrease of cytotoxicity of saporin (data not shown). We further observed that the effect of SA on saporin toxicity could not be enhanced by higher concentrations of SA. It followed an “all or none” principle. Therefore it seems to be a kind of SA-induced transient change of the membrane properties which lead to a sensitization against saporin. It was further evidenced that internalization of saporin most likely occurs via clathrin-mediated endocytosis, followed by the release of saporin into the endosomal transport system and toxin release into the cytosol. Synergistic cytotoxicity between saporin and SA is therefore not a consequence of the generally postulated surface activity of saponins, leading to the formation of pores in the cell membrane [6,37]. However, it is conceivable that SA play a role as a kind of anchor by association with membrane components like cholesterol. This would lead to the insertion of SA molecules into the plasma membrane. The remaining sugar tails could interact with specific endocytic factors, followed by the induction of endocytosis of receptor bound saporin. Enhancement of cytotoxicity of saporin by SA was shown in several malignant cell types formerly derived from the lung (H-1217), brain (SK-N-SH), bladder (ECV304), primary immune system (THP-1, HL-60) and secondary immune system (MOLT-3). Since we have shown that SA trigger clathrin-mediated endocytosis of saporin, we assumed that this would also toke place in normal cells. An increased saporin internalization would lead to strong cytotoxicity in normal cells which limits the use of saporin/SA for therapeutic use. Conflict of interest None. Acknowledgement Funding: German Research Foundation (FU 408/3-1).
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