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Molecular Cancer Therapeutics 6, 782-788, February 1, 2007. doi: 10.1158/1535-7163.MCT-06-0370
© 2007 American Association for Cancer Research
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Research Articles: Therapeutics, Targets, and Development

HIV-Tat protein transduction domain specifically attenuates growth of polyamine deprived tumor cells

Katrin Mani1, Staffan Sandgren3, Johanna Lilja3, Fang Cheng1, Katrin Svensson3, Lo Persson2 and Mattias Belting3

1 Sections of Glycobiology and 2 Biogenic Amines, Department of Experimental Medical Research; and 3 Section of Oncology, Department of Clinical Sciences, Lund University, Lund, Sweden

Requests for reprints: Mattias Belting, Department of Clinical Sciences, Section of Oncology, Lund University, Barngatan 2:1, SE-221 85, Lund, Sweden. Phone: 46-46178549. E-mail: Mattias.Belting{at}med.lu.se

Abstract

Polyamines are essential for tumor cell growth, and the polyamine pathway represents an attractive target for cancer treatment. Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells; however, the mechanism of polyamine entry into mammalian cells remains poorly defined, although a role for proteoglycans has been suggested. Here, we show that the HIV-Tat transduction peptide, which is known to enter cells via a proteoglycan-dependent pathway, efficiently inhibits polyamine uptake. Polyamine uptake–deficient mutant cells with intact proteoglycan biosynthesis (CHO MGBG) displayed unperturbed HIV-Tat uptake activity compared with wild-type cells, supporting the notion that HIV-Tat peptide interferes with polyamine uptake via competition for proteoglycan binding sites rather than a putative downstream transporter. HIV-Tat specifically inhibited growth of human carcinoma cells made dependent on extracellular polyamines by treatment with the polyamine biosynthesis inhibitor {alpha}-difluoromethylornithine; accordingly, the Tat peptide prevented intracellular accumulation of exogenous polyamines. Moreover, combined treatment with {alpha}-difluoromethylornithine and HIV-Tat efficiently blocked tumor growth in an experimental mouse model. We conclude that HIV-Tat transduction domain and polyamines enter cells through a common pathway, which can be used to target polyamine-dependent tumor growth in the treatment of cancer. [Mol Cancer Ther 2007;6(2):782–8]

Introduction

Polyamines and polybasic peptides have been shown to efficiently traverse the plasma membrane barrier of mammalian cells (1, 2). Polybasic peptides, including the HIV-Tat transduction domain, have gained much attention lately due to their ability to carry large cargo (e.g., nucleic acids and proteins) over the cell membrane into the cytoplasm and nuclear compartment of mammalian cells (3, 4). This capacity has been used for introducing therapeutic macromolecules, such as tumor suppressors and proapoptotic proteins, into tumor cells as a possible strategy to treat cancer (5–7). The uptake mechanism of HIV-Tat and other transduction peptides has been extensively studied, and both clathrin- and caveolae-mediated endocytosis and membrane raft-dependent macropinocytosis have been suggested as the major entry pathway (8–12). Moreover, heparan sulfate proteoglycans seem to have an important role in HIV-Tat uptake (1, 2, 11).

Polyamines are essential for cellular proliferation, and elevated polyamine levels have been associated with cell transformation and cancer disease (13–15). Accordingly, depletion of intracellular polyamines by {alpha}-difluoromethylornithine, a specific inhibitor of ornithine decarboxylase that catalyses the first step in the polyamine pathway, results in efficient growth inhibition of tumor cells in vitro and, in some cases, reversal of cell transformation and tumorigenesis in animal models and cancer patients (14–16). Although {alpha}-difluoromethylornithine is still under investigation in clinical antitumor trials (17), the clinical outcome has been overall disappointing due to compensatory recruitment of extracellular polyamines from the circulation by {alpha}-difluoromethylornithine–treated cancer cells in vivo (14). Thus, successful antitumor therapy directed at the polyamine system requires efficient inhibition of both de novo polyamine synthesis and uptake of polyamines from the extracellular milieu.

Several polyamine transport proteins have been cloned and characterized in bacteria and yeast cells, but attempts to find similar transporters in mammalian cells have, thus far, been unsuccessful (18–22). Previous studies from our group have shown that heparan sulfate proteoglycans facilitate spermine uptake, and that combined inhibition of polyamine and heparan sulfate biosynthesis attenuates tumor growth (23, 24). These and other findings identify cell surface heparan sulfate proteoglycans as an attractive target in the treatment of malignant disease (25).

Here, we have explored the possibility that the structurally diverse HIV-Tat transduction peptide and polyamines share a common transport mechanism in human carcinoma cells and that HIV-Tat may be an efficient inhibitor of polyamine uptake and polyamine-dependent tumor growth in vivo.

Materials and Methods

Chemicals and Reagents
Polyamines, ornithine, and fine grade chemicals were from Sigma Chemical Co. (St. Louis, MO). [14C]polyamines and [3H]ornithine were from Amersham International (Bucks, United Kingdom). HIV-Tat peptide (GRKKRRQRRRPPQC), either unlabeled or labeled at the C terminus with AlexaFluor-647, was synthesized by Innovagen AB (Lund, Sweden). Heparan sulfate preparations were the same as previously described (26). {alpha}-Difluoromethylornithine was from Ilex Oncology (San Antonio, TX). Cell media and supplements were from Chemicon Europe, Ltd. (Hampshire, United Kingdom). HiTrap heparin column was from Pharmacia-Amersham.

Cell Culture
Human T24 bladder carcinoma cells were provided by Prof. Inge Ohlsson (Lund University, Lund, Sweden) and cultured in DMEM. Chinese hamster ovary (CHO)-K1 cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in F12K nutrient mixture. Routine culture was done in a humidified 5% CO2 incubator at 37°C, using the respective media supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 µg/mL streptomycin (growth medium).

Heparin Affinity Chromatography of Tat and Spermine
Samples (100 µg Tat, 0.5 mg spermine supplemented with 0.1 µCi [14C]spermine, or 0.5 mg ornithine supplemented with 0.1 µCi [3H]ornithine) were dissolved in 0.5 mL of buffer A (0.1 mol/L Tris, 0.01 mol/L citrate, pH 7.4) and applied to the heparin affinity column, which was eluted using a gradient ranging from 15% to 100% of buffer B (2 mol/L NaCl in buffer A). The effluent was collected in 0.5-mL fractions, and eluted radioactive [14C] and [3H] species were analyzed by liquid scintillation counting. In the case of Tat peptide, the elution profile was registered by an online UV cell measuring absorbance at 215 nm.

Tat Peptide and Polyamine Uptake Experiments
AlexaFluor-647–labeled Tat peptide (0.1 or 1 µg/mL) in serum-free medium was added to extensively rinsed subconfluent cells, and incubation was allowed to proceed for 20 min at 37°C. To exclude the presence of cell surface–associated peptide, cells were extensively rinsed before flow cytometry measurements: three washes with cold PBS supplemented with 100 µg/mL dextran sulfate (molecular weight, 500,000 kDa); trypsination and blocking with 10% FCS; and two consecutive wash/centrifugation cycles using PBS with 1% bovine serum albumin. Cells were finally suspended in PBS with 1% bovine serum albumin and analyzed for fluorescence in a FACS-Calibur (BD Biosciences, San Jose, CA) instrument integrated with Cell-Quest software.

For polyamine uptake experiments, T24 cells were plated in 24-well culture plates at 4 x 104 per well in growth medium, with or without 5 mmol/L {alpha}-difluoromethylornithine. Cells were grown for 3 days to subconfluency, after which varying concentrations of polyamines and [14C]polyamines (specific activity, 31 Ci/mol) in serum-free MEM was added, and then cells were incubated for 20 min at 4°C or 37°C. The incubation medium was removed, and the cells were rinsed with cold MEM containing 1 mmol/L of unlabeled polyamine and then once with MEM and lysed with 0.5 mol/L NaOH for 1 h at 37°C. An aliquot of the cell homogenate was neutralized with 0.5 mol/L HCl and then analyzed for radioactivity by scintillation counting. The difference in cell-associated polyamine levels obtained at 37°C and 4°C was defined as temperature-dependent polyamine internalization.

In vitro Proliferation Assays
The [3H]thymidine incorporation assay was used to assess DNA synthesis. T24 cells were seeded into 24-well culture plates at 70,000 per well in growth medium. After 4 h, the cells were placed in serum-free medium supplemented with insulin (10 ng/mL) and transferrin (25 ng/mL) and allowed to proliferate in the absence or presence of 5 mmol/L {alpha}-difluoromethylornithine for 72 h. The growth medium was then supplemented with 0.5 µmol/L spermidine and/or 100 µg/mL HIV-Tat, and cells were incubated for another 16 h in the presence of 3 µCi/mL [3H]thymidine. The cells were then washed with PBS, after which proteins were precipitated with 10% cold trichloroacetic acid for 30 min. DNA from cell extracts was obtained by incubation in 0.5 mol/L NaOH, 0.5% SDS for 5 min at room temperature, and the amount of incorporated [3H]thymidine was determined by liquid scintillation.

Polyamine Analysis
T24 cells were seeded into 75-cm2 flasks and grown for 72 h in the presence or absence of 5 mmol/L {alpha}-difluoromethylornithine, and growth media were supplemented as described in the appropriate figure legend. After another growth period of 16 h, cells were collected either by scraping or trypsinization, counted and pelleted at 900 x g for 10 min at 4°C and the pellets were stored at –80°C until analysis. Polyamine content in cell pellets extracted with 0.2 mol/L perchloric acid was determined by HPLC (Hewlett Packard 1100; Global Medical Instrumentation, Inc., Ramsey, MN) with o-phthaldialdehyde as the reagent (27).

Metabolic Labeling and Isolation of Proteoglycans
Proteoglycans were metabolically labeled in confluent 75-cm2 cell culture flasks using a sulfate-poor medium supplemented with 50 µCi/mL [35S]sulfate as described previously (28, 29). After 24 h of incubation, the cell layer was extracted with Triton X-100. Proteoglycan material was recovered by passage over DEAE-cellulose. Size separation of proteoglycans and various degradation products thereof was done on a Superose 6 column (Amersham Biosciences, AB, Uppsala, Sweden). Fractions containing heparan sulfate proteoglycan were pooled and further analyzed by ion exchange chromatography on MonoQ (linear gradient elution with 0.3, 1.2, and 2 mol/L NaCl in 7 mol/L urea, pH 8.0; 0.1% Triton X-100). Alternatively, the proteoglycan material obtained from the DEAE-cellulose was analyzed for polyamine affinity on a spermine-substituted HiTrap column (linear gradient elution with 0.15, 1.2, and 2 mol/L NaCl in 10 mmol/L Tris, pH 7.4). Gradients are depicted in respective figures.

Animal Experiments and Tumor Formation In vivo
Female SCIDnodCA mice were kept under pathogen-free conditions in the animal barrier facility at the Biomedical Center, Lund University, in accordance with the Swedish guidelines for humane treatment of laboratory animals. The experimental setup was approved by the ethical committee for animal research in Malmö/Lund, Sweden. For assessment of antitumor activity, human bladder carcinoma T24 cells (1 x 106 in 200 µL PBS) were injected s.c. in the dorsal region of 7- to 8-week-old mice (N = 40). The animals were then randomly divided into control and treatment groups and received (a) no additives, (b) {alpha}-difluoromethylornithine (1%, w/v) via drinking water, (c) daily i.p. injections (0.2 mL) of HIV-Tat (1 µg/µL), or (d) {alpha}-difluoromethylornithine (1%, w/v) and daily i.p. injections (0.2 mL) of HIV-Tat (1 µg/µL). Animals were sacrificed, and tumor mass was determined after 18 days of treatment.

Results

HIV-Tat Peptide Is an Efficient Inhibitor of Polyamine Uptake in Human Carcinoma Cells
Previous work investigated the binding kinetics of growth-promoting polyamines to heparan sulfate, demonstrating Kd of 37 and 110 µmol/L for the interaction of the polyamine spermine with high-sulfated heparan sulfate and low-sulfated heparan sulfate, respectively (30). As a comparison, Kd of HIV-Tat binding to high-sulfated heparan sulfate was reported to be in the low-nanomolar range (31). Accordingly, Tat peptide displayed stronger binding to a heparin (high-sulfated heparan sulfate) affinity column compared with spermine (Fig. 1 ). Tat eluted as a major peak at ~1.4 to 1.5 mol/L NaCl, which is in agreement with previous studies (31, 32). Independent studies have implicated a role for heparan sulfate proteoglycan in the uptake of polyamines and HIV-Tat peptide in CHO cells (1); CHO cell mutants deficient in heparan sulfate proteoglycan showed reduced uptake of the polyamine spermine and HIV-Tat (24, 33). We next investigated whether these structurally unrelated polybasic compounds share the same route of entry in human carcinoma cells. Polyamine biosynthesis inhibition by {alpha}-difluoromethylornithine results in a severalfold induction of polyamine uptake in tumor cells, as has been previously shown (Fig. 2A ; ref. 34). Intriguingly, {alpha}-difluoromethylornithine–treatment also increased HIV-Tat uptake (Fig. 2B). Internalization of both spermidine and spermine was efficiently inhibited by HIV-Tat peptide in a dose-dependent manner (Fig. 2C and D). HIV-Tat was a more efficient inhibitor of spermidine uptake than of spermine uptake (Fig. 2C), which may be expected due to the fact that spermidine and spermine have three and four positive charges, respectively, at physiologic conditions. Importantly, HIV-Tat peptide also showed substantial inhibition of polyamine uptake in {alpha}-difluoromethylornithine–treated cells (Fig. 2D). Consistent with the heparin binding data (Fig. 1), spermine was a poor inhibitor of HIV-Tat peptide uptake (Fig. 2B).


Figure 1
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  		Figure 1. Tat peptide and polyamine binding to heparin. Spermine (0.5 mg) supplemented with 0.1 µCi [14C]spermine or ornithine (0.5 mg) supplemented with 0.1 µCi [3H]ornithine (A) or 100 µg Tat peptide (B) was added to a heparin-substituted HiTrap column, which was eluted with an NaCl gradient, as described in Materials and Methods. Liquid scintillation counting (A) or online UV detection at A215 (B) was done to determine the elution profile of the respective molecules. Data of two independent experiments.

 

Figure 2
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  		Figure 2. HIV-Tat efficiently inhibits polyamine uptake in mammalian cells. A, dose-dependent uptakes of [14C]spermidine (triangles) and [14C]spermine (squares) were determined in T24 cells after preincubation in the absence (filled symbols) or presence (open symbols) of 5 mmol/L {alpha}-difluoromethylornithine for 3 d. B, uptake of AlexaFluor-647–labeled Tat peptide (0.1 or 1 µg/mL) in either untreated (open columns) or {alpha}-difluoromethylornithine–treated (gray columns) T24 cells as in A. In some cases, spermine was added at a 1000-fold (w/w) excess (1 + S). C, the effect of Tat peptide on the uptake of 5 µmol/L [14C]spermine or 5 µmol/L [14C]spermidine in T24 cells. D, same experiment as in C after pretreatment of cells with 5 mmol/L {alpha}-difluoromethylornithine for 3 d. Points, mean (n = 5); bars, SD.

 
The well-established polyamine uptake–deficient CHO-MGBG cell line was isolated as a CHO cell subclone insensitive to methylglyoxal bis(guanylhydrazone) (a highly toxic polyamine analogue that enters cells via the polyamine transport pathway; refs. 18, 35). CHO-MGBG cells showed no alterations in cell-associated proteoglycan production compared with wild-type CHO cells, neither in terms of overall charge density (cf. left and right panels in Fig. 3C ) nor in polyamine affinity (cf. left and right panels in Fig. 3D) as determined by ion exchange and spermine affinity chromatography, respectively. There were no obvious differences in the relative amounts of intact proteoglycans and low molecular weight oligosaccharides (cf. left and right panels in Fig. 3A). Moreover, CHO-MGBG cells displayed intact HIV-Tat peptide internalization compared with wild-type cells (Fig. 3A). Together with the fact that polyamine uptake and HIV-Tat uptake are reduced in proteoglycan-deficient cells (24, 33), our data suggest that HIV-Tat inhibits polyamine uptake via competition for proteoglycan binding sites rather than a putative polyamine-specific transporter that is defective in CHO-MGBG cells.


Figure 3
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  		Figure 3. Polyamine uptake–deficient CHO-MGBG cells exhibit normal proteoglycan production and unperturbed HIV-Tat peptide uptake. A, wild-type CHO-K1 cells and mutant and polyamine uptake–deficient CHO-MGBG cells were grown to subconfluence. After rinsing, cells were incubated for 20 min with AlexaFluor-647-Tat (1 µg/mL). Intracellular AlexaFluor-647-Tat fluorescence was determined by flow cytometry. Columns, mean (n = 4); bars, SD. B-D, proteoglycans were labeled with [3H]glucosamine (GlcN) and [35S]sulfate in CHO-K1 cells (left panel) or CHO-MGBG cells (right panel), and proteoglycan material was analyzed by gel filtration chromatography on a Superpose 6 column (B). Fractions (18–30) corresponding to the heparan sulfate proteoglycan material were pooled and analyzed by ion exchange chromatography on a MonoQ column eluted with 0.3–1.2 mol/L NaCl gradient (C). Alternatively, [35S]sulfate-labeled proteoglycan material was analyzed by affinity chromatography on a spermine-substituted HiTrap column eluted with 0.3 to 1.2 mol/L NaCl gradient (D). In all cases, aliquots of collected fractions were analyzed for radioactivity by liquid scintillation.

 
HIV-Tat Inhibits the Bioavailability of Extracellular Polyamines
It is well established that depletion of intracellular polyamines by {alpha}-difluoromethylornithine treatment results in inhibition of tumor cell proliferation, unless cells are provided with extracellular polyamines, such as in the in vivo situation where polyamines are provided in the diet, from bacteria in the gut and from a constant efflux of intracellular polyamines (13–15). As expected, T24 carcinoma cells showed decreased DNA synthesis in the presence of {alpha}-difluoromethylornithine, and this inhibition could be overcome by restituting the growth medium with polyamine (Fig. 4A ). Whereas HIV-Tat peptide per se had no effect on DNA synthesis, the peptide counteracted the rescue by extracellular polyamine in {alpha}-difluoromethylornithine–treated cells. The data suggest that the HIV-Tat peptide competes for the bioavailability of extracellular polyamines and that the peptide is unable to replace the intracellular activities of polyamines. Consistent with this notion, the Tat peptide efficiently prevented intracellular accumulation of exogenous spermidine in {alpha}-difluoromethylornithine–treated cells (Fig. 4B). The fact that the spermidine level was even lower in cells treated with {alpha}-difluoromethylornithine and Tat peptide than with {alpha}-difluoromethylornithine alone suggests that HIV-Tat also interferes with the reuptake of polyamines that have leaked into the cell medium.


Figure 4
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  		Figure 4. HIV-Tat peptide inhibits the bioavailability of extracellular polyamines. A, T24 cells were grown in the absence (Control) or presence of 5 mmol/L {alpha}-difluoromethylornithine (DFMO) for 72 h. The growth medium was then supplemented with 0.5 µmol/L spermidine (SPD) and 100 µg/mL HIV-Tat, and cells were incubated for another 16 h in the presence of 3 µCi/mL [3H]thymidine. DNA synthesis was determined by measurement of incorporated [3H]thymidine. B, T24 cells were grown in the absence or presence of 5 mmol/L {alpha}-difluoromethylornithine for 72 h. The growth medium was then supplemented with 0.5 µmol/L spermidine and 100 µg/mL HIV-Tat, and cells were incubated for another 16 h. Finally, cells were analyzed for spermidine content, as described in Materials and Methods. Columns, mean (n = 3–6); bars, SD.

 
Tumor Growth Attenuation by Combined {alpha}-Difluoromethylornithine and HIV-Tat Peptide Treatment
We next investigated the combined effect of {alpha}-difluoromethylornithine and HIV-Tat peptide on in vivo tumor growth (Fig. 5 ). A relatively low {alpha}-difluoromethylornithine dose (1% w/v in the drinking water) had no significant effect on T24 carcinoma cell tumor formation under these conditions. Previous studies have shown that i.p. administration of HIV-Tat peptide has high bioavailability at the systemic level (36). Daily injections of HIV-Tat peptide (10 mg/kg) via this route had no significant effect on tumor formation. However, combined treatment with {alpha}-difluoromethylornithine and HIV-Tat peptide dramatically inhibited tumor growth. Average tumor mass of the control group was 51.9 ± 23.4 mg, whereas average tumor mass in the {alpha}-difluoromethylornithine/Tat group was 4.4 ± 2.2 mg (i.e., a 91.5% reduction in tumor mass). Importantly, no signs of serious adverse effects of the {alpha}-difluoromethylornithine/HIV-Tat peptide combination were observed (e.g., the animal weight was unaltered compared with the controls). Only a few of the tumors in the group receiving combined treatment exceeded a diameter of 1 to 2 mm, and the tumors seemed as avascular pale nodes as opposed to the tumors in the other treatment groups that displayed rich vascularization upon gross examination. These observations were supported by very limited CD31 staining in tumors from animals receiving combined treatment.4


Figure 5
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  		Figure 5. Combined treatment with {alpha}-difluoromethylornithine and Tat peptide attenuates in vivo tumor growth. T24 cells were inoculated s.c. on the dorsal side of female SCID mice. The animals were then randomly divided into control and treatment groups receiving (a) no additives, (b) {alpha}-difluoromethylornithine (1%, w/v) via the drinking water, (c) daily i.p. injections (0.2 mL) of HIV-Tat (1 µg/µL), or (d) {alpha}-difluoromethylornithine (1%, w/v) and daily i.p. injections (0.2 mL) of HIV-Tat (1 µg/µL). After 18 d of tumor inoculation, animals were sacrificed and tumor mass was determined. Horizontal bars, average tumor mass of respective groups. Data were obtained from two independent experiments. P values were calculated using Student's t-test.

 
Discussion

We show that a protein transduction peptide derived from HIV-Tat protein efficiently competes with polyamine uptake in mammalian cells and that combined treatment with the polyamine synthesis inhibitor {alpha}-difluoromethylornithine and HIV-Tat peptide inhibits carcinoma cell growth in vitro and attenuates tumor formation in vivo.

We and collaborators have previously shown that proteoglycans facilitate polyamine uptake in mammalian cells; mutant CHO cells deficient in proteoglycan formation were also deficient in polyamine uptake compared with wild-type cells (24). Moreover, proteoglycan-deficient cells were hypersensitive to {alpha}-difluoromethylornithine treatment and insensitive to rescue by extracellular polyamines, both in vitro and in vivo (24). Indeed, it has been shown that {alpha}-difluoromethylornithine–induced polyamine uptake is associated with structural alterations of heparan sulfate proteoglycan that favor polyamine binding and internalization (23, 37). The fact that {alpha}-difluoromethylornithine also seems to up-regulate Tat peptide internalization (Fig. 2B) suggests a common internalization pathway for polyamines and HIV-Tat peptide. The CHO-MGBG cell line that does not accumulate extracellular polyamines and displays normal proteoglycan production when compared with wild-type CHO cells showed unperturbed uptake of Tat peptide (Fig. 3). The data suggest divergent internalization pathways for HIV-Tat and polyamines downstream of the proteoglycan-dependent internalization step and that additional, yet unknown, transport molecule/s are necessary for efficient polyamine uptake.

It is clear from our data that the Tat peptide is unable to mimic the growth-promoting function of polyamines in {alpha}-difluoromethylornithine–treated cells. The HIV-1 Tat protein, reportedly, has effects that are unrelated to its interaction with the target TAR HIV sequence, and both inhibitory and stimulatory effects of exogenously administered Tat on cellular proliferation have been reported (38–42). In our study, Tat efficiently inhibited growth of {alpha}-difluoromethylornithine–treated cells and prevented intracellular accumulation of exogenous polyamine, suggesting that Tat specifically interferes with the bioavailability of extracellular polyamines.

We show that combined {alpha}-difluoromethylornithine and Tat treatment has profound effects on tumor formation in vivo (Fig. 5). Although the Tat peptide per se did not inhibit tumor formation, it is still possible that the Tat peptide, in the context of polyamine deprivation, exhibits effects in addition to inhibition of polyamine uptake. Because heparan sulfate proteoglycans are known to act as coreceptors for major proangiogenic growth factors, most notably vascular endothelial growth factor and fibroblast growth factor 2 (25), it is conceivable that the Tat peptide exerts antiangiogenic effects via competition with growth factors for heparan sulfate proteoglycan binding, thus, leading to blockage of tumor expansion. This would explain the relatively greater in vivo effects of the {alpha}-difluoromethylornithine/Tat peptide combination compared with the in vitro situation. Notably, it has been shown that in ornithine decarboxylase–overexpressing mice that develop spontaneous skin tumors due to elevated polyamine levels, treatment with {alpha}-difluoromethylornithine resulted in tumor regression through an antiangiogenic effect (43). Ongoing studies in our laboratory aim at investigating the role of polyamines in angiogenesis in more detail.

Interestingly, systemic administration of HIV-Tat peptide shows wide tissue distribution, including to the central nervous system (36), which should make gliomas that have overall poor prognosis and have shown partial response to {alpha}-difluoromethylornithine treatment (17, 44) an interesting target for combined {alpha}-difluoromethylornithine and HIV-Tat peptide treatment.

Footnotes

Grant support: Swedish Cancer Fund, Swedish Research Council, Swedish Foundation for Strategic Research (Forskarskolan för läkemedelsvetenskap [FLÄK]), Medical Faculty, Lund University (Avtal om läkarutbildning och forskning [ALF]), Association for International Cancer Research, Crafoordska Foundation, and Gunnar Nilsson Cancer Foundation (M. Belting).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: K. Mani and S. Sandgren contributed equally to this work.

4 K. Mani and M. Belting, unpublished observation. Back

Received 6/26/06; revised 11/16/06; accepted 12/21/06.

References

  1. Belting M. Heparan sulfate proteoglycan as a plasma membrane carrier. Trends Biochem Sci 2003;28:145–51.[CrossRef][Medline]
  2. Belting M, Sandgren S, Wittrup A. Nuclear delivery of macromolecules: barriers and carriers. Adv Drug Deliv Rev 2005;57:505–27.[CrossRef][Medline]
  3. Derossi D, Chassaing G, Prochiantz A. Trojan peptides: the penetratin system for intracellular delivery. Trends Cell Biol 1998;8:84–7.[CrossRef][Medline]
  4. Joliot A, Prochiantz A. Transduction peptides: from technology to physiology. Nat Cell Biol 2004;6:189–96.[CrossRef][Medline]
  5. Wadia JS, Dowdy SF. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv Drug Deliv Rev 2005;57:579–96.[CrossRef][Medline]
  6. Jiang T, Olson ES, Nguyen QT, Roy M, Jennings PA, Tsien RY. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc Natl Acad Sci U S A 2004;101:17867–72.[Abstract/Free Full Text]
  7. Harada H, Hiraoka M, Kizaka-Kondoh S. Antitumor effect of TAT-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumor cells. Cancer Res 2002;62:2013–8.[Abstract/Free Full Text]
  8. Vendeville A, Rayne F, Bonhoure A, Bettache N, Montcourrier P, Beaumelle B. HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses. Mol Biol Cell 2004;15:2347–60.[Abstract/Free Full Text]
  9. Ferrari A, Pellegrini V, Arcangeli C, Fittipaldi A, Giacca M, Beltram F. Caveolae-mediated internalization of extracellular HIV-1 tat fusion proteins visualized in real time. Mol Ther 2003;8:284–94.[CrossRef][Medline]
  10. Fittipaldi A, Ferrari A, Zoppe M, et al. Cell membrane lipid rafts mediate caveolar endocytosis of HIV-1 Tat fusion proteins. J Biol Chem 2003;278:34141–9.[Abstract/Free Full Text]
  11. Richard JP, Melikov K, Brooks H, Prevot P, Lebleu B, Chernomordik LV. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J Biol Chem 2005;280:15300–6.[Abstract/Free Full Text]
  12. Wadia JS, Stan RV, Dowdy SF. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat Med 2004;10:310–5.[CrossRef][Medline]
  13. Pegg AE. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res 1988;48:759–74.[Abstract/Free Full Text]
  14. Wallace HM, Fraser AV. Inhibitors of polyamine metabolism: review article. Amino Acids 2004;26:353–65.[Medline]
  15. Jänne J, Alhonen J, Pietilä M, Keinänen TA. Genetic approaches to the cellular functions of polyamines in mammals. Eur J Biochem 2004;271:877–94.[Medline]
  16. Gerner EW, Meyskens FL, Jr. Polyamines and cancer: old molecules, new understanding. Nat Rev Cancer 2004;4:781–92.[CrossRef][Medline]
  17. Levin VA, Prados MD, Yung WK, Gleason MJ, Ictech S, Malec M. Treatment of recurrent gliomas with eflornithine. J Natl Cancer Inst 1992;84:1432–7.[Abstract/Free Full Text]
  18. Byers TL, Wechter R, Nuttall ME, Pegg AE. Expression of a human gene for polyamine transport in Chinese-hamster ovary cells. Biochem J 1989;263:745–52.[Medline]
  19. Seiler N, Delcros JG, Moulinoux JP. Polyamine transport in mammalian cells. An update. Int J Biochem Cell Biol 1996;28:843–61.[CrossRef][Medline]
  20. Igarashi K, Kashiwagi K. Polyamine transport in bacteria and yeast. Biochem J 1999;344:633–42.
  21. Aouida M, Leduc A, Poulin R, Ramotar D. AGP2 encodes the major permease for high affinity polyamine import in Saccharomyces cerevisiae. J Biol Chem 2005;280:24267–76.[Abstract/Free Full Text]
  22. Uemura T, Kashiwagi K, Igarashi K. Uptake of putrescine and spermidine by Gap1p on the plasma membrane in Saccharomyces cerevisiae. Biochem Biophys Res Commun 2005;328:1028–33.[CrossRef][Medline]
  23. Belting M, Persson S, Fransson LÅ. Proteoglycan involvement in polyamine uptake. Biochem J 1999;338:317–23.
  24. Belting M, Borsig L, Fuster MM, et al. Tumor attenuation by combined heparan sulfate and polyamine depletion. Proc Natl Acad Sci U S A 2002;99:371–6.[Abstract/Free Full Text]
  25. Fuster MM, Esko JD. The sweet and sour of cancer: glycans as novel therapeutic targets. Nat Rev Cancer 2005;5:526–42.[CrossRef][Medline]
  26. Fransson LÅ, Sjöberg I, Havsmark B. Structural studies on heparan sulphates. Characterization of oligosaccharides; obtained by periodate oxidation and alkaline elimination. Eur J Biochem 1980;106:59–69.[Medline]
  27. Seiler N, Knödgen B. High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives. J Chromatogr 1980;221:227–35.[Medline]
  28. Ding K, Jönsson M, Mani K, Sandgren S, Belting M, Fransson LÅ. N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and nitrite-deprived endothelial cells. Mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein. J Biol Chem 2001;276:3885–94.[Abstract/Free Full Text]
  29. Mani K, Jönsson M, Edgren G, Belting M, Fransson LÅ. A novel role for nitric oxide in the endogenous degradation of heparan sulfate during recycling of glypican-1 in vascular endothelial cells. Glycobiology 2000;10:577–86.[Abstract/Free Full Text]
  30. Belting M, Havsmark B, Jönsson M, Persson S, Fransson LÅ. Heparan sulphate/heparin glycosaminoglycans with strong affinity for the growth-promoter spermine have high antiproliferative activity. Glycobiology 1996;6:121–9.[Abstract/Free Full Text]
  31. Ziegler A, Seelig J. Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters. Biophys J 2004;86:254–63.
  32. Rusnati M, Tulipano G, Spillmann D, et al. Multiple interactions of HIV-I Tat protein with size-defined heparin oligosaccharides. J Biol Chem 1999;274:28198–205.[Abstract/Free Full Text]
  33. Tyagi M, Rusnati M, Presta M, Giacca M. Internalization of HIV-1 tat requires cell surface heparan sulfate proteoglycans. J Biol Chem 2001;276:3254–61.[Abstract/Free Full Text]
  34. Alhonen-Hongisto L, Seppanen P, Jänne J. Intracellular putrescine and spermidine deprivation induces increased uptake of the natural polyamines and methylglyoxal bis(guanylhydrazone). Biochem J 1980;192:941–5.[Medline]
  35. Mandel JL, Flintoff WF. Isolation of mutant mammalian cells altered in polyamine transport. J Cell Physiol 1978;97:335–43.[CrossRef][Medline]
  36. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 1999;285:1569–72.[Abstract/Free Full Text]
  37. Ding K, Sandgren S, Mani K, Belting M, Fransson LÅ. Modulations of glypican-1 heparan sulfate structure by inhibition of endogenous polyamine synthesis. Mapping of spermine-binding sites and heparanase, heparin lyase, and nitric oxide/nitrite cleavage sites. J Biol Chem 2001;276:46779–91.[Abstract/Free Full Text]
  38. Pugliese A, Vidotto V, Beltramo T, Petrini S, Torre D. A review of HIV-1 Tat protein biological effects. Cell Biochem Funct 2005;23:223–7.[CrossRef][Medline]
  39. Gibellini D, Vitone F, Schiavone P, Re MC. HIV-1 tat protein and cell proliferation and survival: a brief review. New Microbiol 2005;28:95–109.[Medline]
  40. Bettaccini AA, Baj A, Accolla RS, Basolo F, Toniolo AQ. Proliferative activity of extracellular HIV-1 Tat protein in human epithelial cells: expression profile of pathogenetically relevant genes. BMC Microbiol 2005;5:20.[CrossRef][Medline]
  41. Lotz M, Clark-Lewis I, Ganu V. HIV-1 transactivator protein Tat induces proliferation and TGF ß expression in human articular chondrocytes. J Cell Biol 1994;124:365–71.[Abstract/Free Full Text]
  42. Conaldi PG, Bottelli A, Baj A, et al. Human immunodeficiency virus-1 tat induces hyperproliferation and dysregulation of renal glomerular epithelial cells. Am J Pathol 2002;161:53–61.[Abstract/Free Full Text]
  43. Lan L, Trempus C, Gilmour SK. Inhibition of ornithine decarboxylase (ODC) decreases tumor vascularization and reverses spontaneous tumors in ODC/Ras transgenic mice. Cancer Res 2000;60:5696–703.[Abstract/Free Full Text]
  44. Levin VA, Hess KR, Coucair A, et al. Phase III randomized study of postradiotherapy chemotherapy with combination {alpha}-difluoromethylornithine-PCV versus PCV for anaplastic gliomas. Clin Cancer Res 2003;9:981–90.[Abstract/Free Full Text]




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