Generation of New Cytotoxic Human Ribonuclease ... - ACS Publications

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Generation of New Cytotoxic Human Ribonuclease Variants Directed to the Nucleus Anna Vert, Jessica Castro, Santiago Ruiz-Martínez, Pere Tubert, Diego Escribano, Marc Ribó, Maria Vilanova,* and Antoni Benito* Laboratori d’Enginyeria de Proteïnes, Departament de Biologia, Facultat de Ciències, Universitat de Girona, Campus de Montilivi, M. Aurélia Campmany, 69 17071 Girona, Spain, and Institut d’Investigació Biomèdica de Girona Dr. Josep Trueta (IdIBGi), Girona, Spain ABSTRACT: Ribonucleases are promising agents for use in anticancer therapy. Engineering a nuclear localization signal into the sequence of the human pancreatic ribonuclease has been revealed as a new strategy to endow this enzyme with cytotoxic activity against tumor cells. We previously described a cytotoxic human pancreatic ribonuclease variant, named PE5, which is able to cleave nuclear RNA, inducing the apoptosis of cancer cells and reducing the amount of P-glycoprotein in different multidrugresistant cell lines. These results open the opportunity to use this ribonuclease in combination with other chemotherapeutics. In this work, we have investigated how to improve the properties of PE5 as an antitumor drug candidate. When attempting to develop a recombinant protein as a drug, two of the main desirable attributes are minimum immunogenicity and maximum potency. The improvements of PE5 have been designed in both senses. First, in order to reduce the potential immunogenicity of the protein, we have studied which residues mutated on PE5 can be reverted to those of the wild-type human pancreatic ribonuclease sequence without affecting its cytotoxicity. Second, we have investigated the effect of introducing an additional nuclear localization signal at different sites of PE5 in an effort to obtain a more cytotoxic enzyme. We show that the nuclear localization signal location is critical for the cytotoxicity. One of these variants, named NLSPE5, presents about a 10-fold increase in cytotoxicity respective to PE5. This variant induces apoptosis and kills the cells using the same mechanism as PE5. KEYWORDS: human pancreatic ribonuclease, nuclear delivery, cytotoxicity, protein engineering, antitumor drug

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localization signal (NLS) which is recognized by α-importin.16 This NLS targets the protein to the nucleus, specifically into the nucleolus,15 where RI is absent. In contrast to other cytotoxic RNases, PE5 cleaves nuclear RNA while leaving cytoplasmatic RNA unaffected.17 As expected, the effects on gene expression and the mechanism of cytotoxicity of this variant are different from those presented by other cytotoxic RNases like onconase.18 Interestingly, PE5 reduces the expression level of P-glycoprotein (P-gp) on multidrug-resistant cell lines (MDR).19 In the present work we have designed strategies to improve the efficiency of PE5 as an antitumor drug candidate. First, in order to obtain a more cytotoxic enzyme we have investigated the effect of introducing a new NLS on PE5 and we show that the site of insertion is critical for its cytotoxicity. Among the different constructions assayed, NLSPE5, which carries the NLS of SV40 large T-antigen at its N-terminus, is as cytotoxic as onconase for a panel of different tumor cell lines. And second, in an effort at reducing the potential immunogenicity of

INTRODUCTION Members of the pancreatic ribonuclease (RNase) superfamily display an array of biological activities, some of which are promising agents for use in anticancer therapy.1 Among them, the case of onconase, a monomeric RNase isolated from Rana pipiens (northern leopard frog), manifests cytotoxic and cytostatic effects,2 presents synergism with several kinds of anticancer drugs,3−10 and at present is in phase II/III of clinical trials as an anticancer drug.11 Onconase, nevertheless, shows renal toxicity at high concentrations.12,13 Therefore the generation of cytotoxic variants of a human RNase such as the human pancreatic RNase (HP-RNase) would undoubtedly provide a potentially useful therapeutic agent which would be expected to have lower immunogenicity and renal toxicity than onconase. Several variants of HP-RNase have been designed to be cytotoxic (for a review see ref 14) because either they are resistant to the cytosolic RNase inhibitor (RI) or they are targeted to tumor cells through specific ligands that ensure an efficient arrival to the cytosol. We previously envisaged a new strategy for constructing cytotoxic RNases consisting of routing them into the nucleus. Formerly, we reported a cytotoxic variant of HP-RNase, named PE5, which, despite being sensitive to the RI, is cytotoxic for a panel of diverse cell lines.15 This variant carries a conformational bipartite nuclear © 2012 American Chemical Society

Received: Revised: Accepted: Published: 2894

April 18, 2012 September 5, 2012 September 7, 2012 September 7, 2012 dx.doi.org/10.1021/mp300217b | Mol. Pharmaceutics 2012, 9, 2894−2902

Molecular Pharmaceutics

Article

Figure 1. Schematic representation of the main HP-RNase variants used in this work. PE5 is an HP-RNase variant that carries a conformational bipartite NLS constituted by the residues dashed in light gray. PE5 differs in seven residues from HP-RNase (positions 4, 6, 9, 16, 17, 89, and 90), whereas PE9 differs in 2 residues (positions 89 and 90) and PE10 in 4 residues (positions 4, 6, 89, and 90). NLSPE5 carries the NLS of SV40 large T-antigen at the N-terminus of PE5, separated by a spacer. PE5NLS and PE5spNLS incorporate the same NLS at the C-terminus of PE5, without or with a spacer between PE5 and the NLS, respectively. The additional NLS of these PE5 variants is shown in dark gray.

RNase Expression and Purification. PE5 variants and onconase were produced and purified from Escherichia coli BL21 (DE3) cells transformed with the corresponding vector as described previously.21−23 The molecular mass of each variant was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the Unitat cientif icotècnica de suport of the Institut de Recerca of the Hospital Universitari Vall d’Hebron (Barcelona, Spain). The protein concentration of each variant was determined by UV spectroscopy using an extinction coefficient of ε280 = 7950 M−1 cm−1 for HP-RNase variants and of ε280 = 10470 M−1 cm−1 for onconase, calculated using the method devised by Pace et al.24 Determination of Thermal Stability. Temperatureunfolding studies were carried out essentially as described previously.25 Depending on the experiment, proteins were dissolved to a concentration of 0.8 mg/mL in 50 mM sodium acetate buffer at pH 5.0 or 100 mM sodium acetate buffer at pH 4.0. Temperature-unfolding transition curves were fitted to a two-state thermodynamic model combined with sloping linear functions for the native and denatured states, and the thermodynamic parameters were calculated as reported previously.26 All data are described as the mean ± standard error (SE) of three independent determinations. Determination of Steady-State Kinetic Parameters. Spectrophotometric assays27 were used to determine the kinetic parameters for the hydrolysis of cytidine 2′,3′-cyclic monophosphate (C>p) by the HP-RNase variants. Steady-state kinetic parameters were obtained by nonlinear regression analysis using the program ENZFITTER (Elsevier Biosoft, U.K.). All data are described as the mean ± standard error (SE) of three determinations. RNase Inhibitor Binding Assay. RNases were tested for ribonucleolytic activity in the presence of RI (Promega, USA) using an agarose gel-based assay as described previously.15 Cell Lines and Culture Conditions. NCI/ADR-RES human ovarian cancer MDR cell line (formerly MCF-7/Adr) was a generous gift from Dr. Ramon Colomer of the Institut Català d’Oncologia de Girona, Hospital Universitari de Girona Dr. Josep Trueta (Girona, Spain); Jurkat human T-cell lymphoblastlike cell line and HeLa human cervical cancer cell line were obtained from Eucellbank (Universitat de Barcelona, Barcelona, Spain); NCI-H460/R human lung cancer MDR cell line28 was a generous gift from Dr. Sabera Ruzdijić of the S. Stanković

PE5 we have studied which residues that differ from those found in wild-type HP-RNase can be reverted to the human sequence without affecting its cytotoxicity. We have therefore constructed a new version of PE5 in which half of the mutated residues (residues 9, 16, and 17) have been replaced by those present in the wild-type enzyme.

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EXPERIMENTAL SECTION RNase Variants. Plasmids expressing PE5 and PE3 (pE5 and pE3) have been described previously.15 PE5 was constructed from PM5 replacing Gly89 and Ser90 by Arg. PE3 was constructed from PM5 replacing Arg31 and Arg91 by Glu and Ala, respectively. PM5 codes for HP-RNase and incorporates the substitutions Arg4 and Lys6 to Ala, Gln9 to Glu, Asp16 to Gly and Ser17 to Asn.20 Variants Carrying an Additional NLS. Variants NLSPE5 and NLSPE3 carry the NLS of SV40 large T-antigen (PKKKRKVE) at the N-terminus of PE5 and PE3, respectively. Variants scNLSPE5 and scNLSPE3 carry a scrambled form of the NLS (KPKERVKKA) at the N-terminus of PE5 and PE3, respectively. In all cases, the basic stretches are linked to the Nterminus of the RNase by a two-residue linker (AS). Variants PE5NLS and PE5scNLS carry the same NLS or its scrambled form, respectively, at the C-terminus of the protein. In variants PE5spNLS and PE5spscNLS, the NLS or scrambled NLS is linked to the C-terminus of the protein by a five-residue linker (SVGGS), respectively. These variants were constructed using cassette mutagenesis strategy and confirmed by DNA sequencing. Figure 1 indicates the modifications introduced in the main variants and also the position of the different NLSs in the sequence. H119A Variants, PE9 and PE10. These variants were created by site-directed mutagenesis using the QuikChange sitedirected mutagenesis kit (Stratagene, USA) and following the manufacturer’s instructions. PE9 was constructed from PM9 (wild-type HP-RNase,20) by exchanging residues Gly89 and Ser90 by Arg. PE10 was constructed from PE9 by replacing residues Arg4 and Lys6 by Ala. Figure 1 indicates the modifications introduced in these two variants and also the residues implicated in the NLS. NLSPE5H119A was constructed from variant NLSPE5 by replacing residue His119 by Ala. All the different constructions were checked by DNA sequencing. 2895

dx.doi.org/10.1021/mp300217b | Mol. Pharmaceutics 2012, 9, 2894−2902


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chromogenic substrates, DEVD-pNA, IETD-pNA, and LEHDpNA, by caspases-3, -8, and -9, respectively. Briefly, NCI/ADRRES cells (1.1 × 106 cells/100 mm dish) were incubated with 4.85 μM NLSPE5 or 44.5 μM PE5 for 24, 48, and 72 h in serum-starved medium. Then, attached and floating cells were harvested at 460g for 10 min at 4 °C, washed twice in cold PBS, lysed, and centrifuged at 10000g for 5 min at 4 °C. The supernatant was recovered, and the protein concentration was determined using the Bradford protein assay (Bio-Rad Laboratories, USA). Afterward, 10 μL of the cell lysate corresponding to 20 μg of total protein, 10 μL of 2X reaction buffer containing 10 mM DTT, and 1 μL of the 10 mM DEVDpNA, IETD-pNA, or LEHD-pNA substrates were mixed. The reactions were then incubated at 37 °C for 4 h. The reaction was measured by changes in absorbance at 405 nm. All data are described as the mean ± standard error (SE) of three determinations. Western Blot Analysis. NCI/ADR-RES cells (9 × 105 cells/100 mm dish) were incubated with 0.97 μM NLSPE5 or 8.9 μM PE5 for 72 h. Treatments produced in all cases a decrease of 50% in cell proliferation (IC50). Quantification of GAPDH, cyclin D1, cyclin E, and p21WAF1/CIP1 was performed by Western blot as previously described.18 The linearity of the assay was preliminarily checked for each monoclonal antibody by submitting different amounts of untreated cell extracts to Western blotting. All data are described as the mean ± standard error (SE) of at least three independent determinations. Doxorubicin Accumulation Assay. Intracellular doxorubicin levels were determined by flow cytometry.19 Briefly, NCI-H460/R cells (105 per well) were seeded into 6-well plates and then treated with NLSPE5 (0.15, 0.35, and 0.50 μM) or PE5 (1.0, 2.6, and 4.3 μM). These concentrations produced a decrease of cell proliferation of 20%, 40%, and 50%, respectively. After 72 h of treatment, the cells were incubated with 10 μM doxorubicin for 1 h at 37 °C in 5% CO2. As control experiments 10 μM verapamil (Sigma-Aldrich, USA), which is a competitive inhibitor of P-gp, was added in combination with the doxorubicin. Data are described as the mean ± standard error (SE) of at least three independent determinations.

Institute for Biological Research (Belgrade, Serbia); NCI-H460 human lung cancer cell line and OVCAR-8 human ovarian cancer cell line were obtained from the National Cancer Institute-Frederick DCTD tumor cell line repository. NCI/ ADR-RES and HeLa cells were routinely grown at 37 °C in a humidified atmosphere of 5% CO2 in DMEM (Gibco, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Germany), 50 U/mL penicillin and 50 μg/mL streptomycin (Gibco, Germany). The other cell lines were grown at 37 °C in a humidified atmosphere of 5% CO2 in RPMI (Gibco, Germany) supplemented with 10% FBS, 50 U/ mL penicillin and 50 μg/mL streptomycin. NCI/ADR-RES and NCI-H460/R cells were maintained in media containing 1.84 μM and 0.1 μM doxorubicin (Tedec-Meijic Farma, Spain), respectively. Cells remained free of Mycoplasma and were propagated according to established protocols. Cell Proliferation Assays. Cells were seeded into 96-well plates at the appropriate density, i.e., 10,000 (for NCI/ADRRES), 1,500 (for OVCAR-8), 3,000 (for NCI-H460/R), 1,900 (for NCI-H460), 1,100 (for HeLa), and 6,000 (for Jurkat) cells. After 24 h of incubation, cells were treated with various concentrations of RNase for 72 h. Drug sensitivity was determined by the MTT method essentially following the manufacturer’s instructions (Sigma, USA) and according to ref 18. All data are described as the mean ± standard error (SE) of at least three independent experiments with three replicas in each. Cytosolic and Nuclear RNA Degradation Assay. 3 × 106 HeLa cells were seeded into T75 flasks. After 4 h of incubation they were treated for 24 h with 0.3 μM NLSPE5 or 1 μM PE5. Treatments produced in all cases a decrease of 10% in cell proliferation. Nuclear and cytoplasmic RNA was extracted using the PARIS kit (Applied Biosystems/Ambion, USA) according to the manufacturer’s instructions. RNA degradation of each sample was quantified in the Scientific Services of the Centre de Regulació Genòmica (Barcelona, Spain) using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RIN values were obtained from the analysis of the electropherograms. The RIN algorithm allows the calculation of RNA integrity using a trained artificial neural network based on the determination of features that can be extracted from the electrophoretic traces. The selected features which collectively catch the most information about the integrity levels include the ratio of area of ribosomal bands to total area of the electropherogram, the height of the 18S peak, the ratio of the area in the fast region to the total area of the electropherogram, and the height of the lower marker. The output RIN is a decimal or integer number in the range of 1−10: a RIN of 1 is returned for a completely degraded RNA sample whereas a RIN of 10 is achieved for intact RNA samples. Phosphatidylserine Exposure Assay. Quantitative analysis of NCI/ADR-RES apoptotic cell death caused by treatment with 4.85 μM NLSPE5 or 44.5 μM PE5 was performed by flow cytometry using the Alexa Fluor 488 annexin V/PI Vybrant Apoptosis Assay Kit (Molecular Probes, USA) following the manufacturer’s instructions and according to ref 18. These RNase concentrations corresponded to five times the IC50 after 72 h of incubation. All data are described as the mean ± standard error (SE) of at least three independent experiments. Caspase Activation Assay. Caspase-3, -8, and -9 catalytic activities were measured using the APOPCYTO caspase-3, -8, and -9 colorimetric assay kits (MBL, Japan) following the manufacturer’s protocol. The assay is based on cleavage of the

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RESULTS Back-Mutation of PE5 To Obtain a More Human Sequence. We were interested in reducing the potential immunogenicity of PE5. To this end, we studied which PE5 residues different from those of wild-type HP-RNase could be reverted to the human sequence without affecting its cytotoxicity. First we constructed PE9 variant, in which the N-terminal residues mutated in PE5 (positions 4, 6, 9, 16, and 17), which are not important for the NLS, were reverted to those of the wild-type enzyme (Figure 1). As a result, this variant still carries the residues that constitute the NLS of PE5, and it was expected that it exhibited the same cytotoxicity. Surprisingly, when its cytotoxicity was measured in the NCI/ ADR-RES cell line, PE9 was clearly less cytotoxic than PE5 (Table 1). The five substitutions did not change the catalytic efficiency of the variant in comparison to PE5 but decreased its thermal stability by about 4 °C (Table 1). This latter result was expected since we had previously described that these five replacements present in PE5 increased the thermal stability of the HP-RNase by 4 °C.29 Among them, replacements of Arg4 and Lys6 by Ala mainly contributed to the increase in stability. We also constructed PE10, a PE9 variant in which residues Arg4 and Lys6 were replaced by Ala (Figure 1). The 2896



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The Introduction of an Additional NLS May Increase the Cytotoxicity of PE5 Depending on the Insertion Site. In order to increase the ability of PE5 to reach the nucleus we designed two new variants, NLSPE5 and PE5NLS, by inserting the sequence of the NLS of SV40 large T-antigen either at the N- or C-terminus of PE5, respectively (Figure 1). This sequence had been previously used to direct green fluorescent protein inside the nucleus.30 In order to exclude chargedependent effects, we also created two control variants (scNLSPE5 and PE5scNLS) carrying a scrambled NLS, that is, a non-NLS sequence with the same amino acid composition, at either the N- or C-terminus of PE5. We checked that this scrambled sequence was not an NLS using the protein analysis tool PredictNLS (http://cubic.bioc.columbia.edu/predict NLS/). We then measured the thermal stability and the catalytic efficiency of these variants (Table 2). For some of the variants

Table 1. Biochemical Characterization of PE5 Variants BackMutated to HP-RNase

PE5 PE9 PE10

replacementsa

Tmb (°C)

kcat/Kmc (%)

IC50d (μM)

R4A, K6A, Q9E, D16G, S17N, G89R, S90R G89R, S90R R4A, K6A, G89R, S90R

45.4 ± 0.1

100.0 ± 0.0

8.9 ± 0.5

41.1 ± 0.1 45.5 ± 0.1

126.6 ± 0.6 128.9 ± 6.2

>30e 8.9 ± 1.1

a

Replacements in relation to HP-RNase. Replacements Gly89Arg and Ser90Arg (bold) are part of the NLS of the RNases. bMeasured at pH = 5.0 (mean ± SE). cCatalytic efficiency of hydrolysis of C>p relative to that of PE5 (mean ± SE). dMeasured for NCI/ADR-RES cell line (mean ± SE). eThe IC50 was not reached under the tested conditions.

cytotoxicity of this new variant was equal to that of PE5 (Table 1) and was not restricted to the NCI/ADR-RES cell line (not shown). Also, the thermal stability, the catalytic efficiency (Table 1), and the ability to evade the RI (Figure 2) were similar to those of PE5. We have therefore obtained a cytotoxic PE5 variant in which half of the residues that differed from wild-type HP-RNase are back-mutated.

Table 2. Biochemical Characterization of PE5 Variants Carrying an Additional NLS or a Scrambled NLS Sequence PE5 NLSPE5 scNLSPE5 PE5NLS PE5scNLS PE5spNLS PE5spscNLS

Tma (°C)

kcat/Kmb (%)

IC50c (μM)

± ± ± ± ± ± ±

100.0 ± 0.0 84.4 ± 2.5 83.7 ± 2.2 −d −d 93.2 ± 3.0 110.5 ± 0.6

8.9 ± 0.5 1.0 ± 0.1 2.3 ± 0.1 NDe NDe 10.6 ± 0.9 7.7 ± 0.2

36.6 35.8 35.9 23.5 23.8 35.1 35.1

0.1 0.1 0.1 0.1 0.1 0.1 0.3

Measured at pH = 4.0 (mean ± SE). bCatalytic efficiency of hydrolysis of C>p relative to that of PE5 (mean ± SE). cMeasured for NCI/ADR-RES cell line (mean ± SE). dThe catalytic activity was undetectable at the higher concentration of substrate used. eNot done. a

we could not calculate the Tm at pH 5.0 because they presented a nonreversible denaturation process that precluded the determination of the Tm. Different pHs were checked (not shown), and it was observed that at pH 4.0 the unfolding was fully reversible. Pancreatic RNase’s stability decreases at acidic pHs,31 and Tm values obtained at pH 4.0 are therefore underestimating the stability at neutral pH. Accordingly, for those variants whose unfolding was reversible at pH 5.0 their Tm at this pH increased about 8.5 °C respective to the value obtained at pH 4.0 (not shown), well over the physiological temperature. While the Tm of NLSPE5 and scNLSPE5 was similar to that of PE5, the Tm of PE5NLS and PE5scNLS was clearly lower (Table 2). We also checked the catalytic efficiency of these variants using C>p as a substrate. The catalytic efficiency of NLSPE5 and scNLSPE5 was equivalent to that of PE5, but we could not detect the catalytic activity of PE5NLS and PE5scNLS even at the higher substrate concentration used (Table 2). We postulated that the NLS basic sequence at the Cterminus of the protein was interfering somehow with its structure, and we decided not to carry out further analysis with PE5NLS and PE5scNLS. We constructed therefore two new variants (PE5spNLS and PE5spscNLS) that carried an additional spacer (SVGGS) between PE5 and the NLS (Figure 1). The Tm and the catalytic efficiency of these two new variants were nearly equal to those of PE5 (Table 2). We assayed the cytotoxicity of the RNase variants against the NCI/ADR-RES cell line (Table 2). NLSPE5 had an IC50 clearly lower than its control scNLSPE5 and than PE5. PE5spNLS and PE5spscNLS were as cytotoxic as the parental PE5. The higher

Figure 2. Inhibition of ribonucleolytic activity by the RI. Inhibition was assessed by comparing on an agarose gel the RNase catalyzed degradation of 16S and 23S rRNA catalyzed by 15 ng of RNase in the presence (+) and absence (−) of 40 units of the RI (40 U of RI can inhibit about 100 ng of bovine RNase A, i.e., 6-fold excess of RI over the RNase constructs). ONC represents onconase, and C corresponds to the control assay without RNase. As ribonucleolytic activity in the assay decreases, both band intensities and apparent molecular masses increase. (A) RI evasion assay of PE10, PE5, and onconase. (B) RI evasion assay of RNases carrying an additional NLS. 2897



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characterization of the most cytotoxic variant, NLSPE5, we investigated whether the ribonucleolytic activity was essential for its cytotoxicity. Consequently, we abolished the ribonucleolytic activity of NLSPE5 by creating a new variant in which His119 catalytic residue was substituted by Ala. This variant was neither catalytically active nor cytotoxic (not shown), indicating that ribonucleolytic activity is essential for the cytotoxicity of NLSPE5. The interaction of the cytotoxic variants with the RI could be a critical factor in determining the cytotoxic potency. Therefore, RI inhibition was qualitatively investigated in an agarose gel-based assay. All the variants were fully inhibited by the RI (Figure 2), but NLSPE5 showed a slight RNA degradation. This result could indicate that the increase in cytotoxicity of NLSPE5 could be caused by the acquisition of the ability to evade the RI and cleave cytosolic RNA. To check this possibility, we investigated the integrity of the nuclear and cytoplasmic RNA of HeLa cells treated with NLSPE5. We incubated HeLa cells with 0.3 μM NLSPE5 or 1 μM PE5 for 24 h. The conditions were chosen because they correspond to the minimal incubation time and concentrations necessary to observe a cytotoxic effect (inhibition of 10% of cell growth) and therefore it can be considered that the RNA degradation is due to the direct action of the RNase but not to the induction of cellular apoptosis.32 RNA degradation was quantified using a bioanalyzer (Figure 5). Cytoplasmatic RNA was not degraded

cytotoxicity of NLSPE5 was not restricted to this cell line. Figure 3 shows that the degree of cytotoxicity of NLSPE5 is

Figure 3. Cytotoxicity of RNase variants for different cell lines. IC50 values of onconase (black bars), NLSPE5 (striped bars), and PE5 (checkered bars) for the indicated tumor cell lines. Control and RNase-treated cell cultures were maintained for 72 h, and metabolic activity was determined by MTT assay as described in the Experimental Section.

similar to that of onconase and 6 to 14 times higher than that of PE5 in a large panel of cell lines representative of different human cancers. The IC50 for MDR cell lines (NCI/ADR-RES and NCI-H460/R) was higher than for their respective parental cell lines (OVCAR-8 and NCI-H460). Hence, we had obtained a much more cytotoxic HP-RNase variant, NLSPE5, by inserting an additional NLS at the N-terminus of PE5. We were interested to know whether the N-terminal insertion of the NLS would promote the cytotoxicity of other RNase variants. Therefore, we attached the NLS to the Nterminus of wild-type HP-RNase variant PE315 to create NLSPE3. In PE3 Arg31 and Arg91 are substituted by Gly and Ala, respectively. PE3 has not an NLS, and although it evades the RI, it has a low cytotoxicity. The inclusion of the NLS at its N-terminus produced a clear increase of its cytotoxicity (Figure 4) although it was 2-fold lower than that of PE5. Again, when a scrambled sequence was inserted at the same site to create scNLSPE3, the increase in cytotoxicity was much lower. The Cytotoxicity of NLSPE5 Is Dependent on the Cleavage of Nuclear RNA. As a first stage in the

Figure 5. Assessment of in vivo nuclear and cytoplasmatic RNA degradation in HeLa cells incubated with 0.3 μM NLSPE5 or 1 μM PE5. C corresponds to untreated cells. RNA degradation was analyzed using Agilent 2100 Bioanalyzer electropherogram profiles of RNA purified samples obtained after incubation of HeLa cells with the indicated RNases for 24 h.

at all in the different samples while nuclear RNA degradation was evident for NLSPE5- and PE5-treated cells but not for untreated cells. Nuclear RNA cleavage induced by NLSPE5 was dose-dependent since incubation with 0.3 μM produced an RNA integrity number (RIN) of 4.5 whereas a lower dose (0.1 μM) produced a RIN of 6.5 (not shown). The Effects Induced by NLSPE5 on Cancer Cells Are Analogous to Those of PE5. We investigated whether the cytotoxic properties of NLSPE5 differed from those of PE5. First, we quantified by FACS analysis the percentage of NCI/ ADR-RES cells in early and late apoptosis and necrosis after 24, 48, and 72 h of incubation with 4.85 μM NLSPE5 or 44.5 μM PE5. In all cases early apoptosis was evident after 24 h of treatment whereas an important fraction of cells were in late apoptosis after 48 h of treatment (Table 3). The variants did

Figure 4. Effect of different concentrations of PE3 (□), NLSPE3 (●), and scNLSPE3 (○) on NCI/ADR-RES cell growth. Control and RNase-treated cell cultures were maintained for 72 h, and metabolic activity was determined by the MTT assay as described in the Experimental Section. Cell growth is expressed as the percentage of control activity using the absorbance values. Curves in the figure are from one representative experiment. Equivalent results were found in at least three independent experiments. 2898



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Table 3. Apoptosis Induced by NLSPE5 and PE5 Measured by Annexin V-Alexa Fluor 488/PI Staininga control 24 h early apoptotic cells (%) late apoptotic cells (%) necrotic cells (%) viable cells (%)

7.1 7.3 1.3 84.3

± ± ± ±

48 h 0.3 0.7 0.1 1.0

5.3 7.8 1.4 85.5

± ± ± ±

NLSPE5 72 h

0.2 0.4 0.1 0.3

5.6 9.2 1.3 83.9

± ± ± ±

24 h 0.2 0.2 0.2 0.5

18.2 8.7 1.2 71.9

± ± ± ±

48 h 0.6 0.6 0.2 0.3

31.8 17.1 0.7 50.4

± ± ± ±

PE5 72 h

1.0 1.8 0.2 0.6

41.0 31.0 0.5 27.5

± ± ± ±

24 h 1.0 2.1 0.1 1.7

15.2 7.3 1.1 76.4

± ± ± ±

48 h 0.1 1.1 0.1 0.9

28.6 16.4 0.7 54.3

± ± ± ±

72 h 2.5 0.6 0.1 1.9

45.8 30.9 0.3 23.0

± ± ± ±

3.7 1.2 0.1 2.4

a NCI/ADR-RES cells were treated with 4.85 μM NLSPE5 or 44.5 μM PE5 for 24, 48, or 72 h. Cells undergoing early apoptosis were positive for Annexin V and negative for PI (Annexin V+/PI−), late apoptotic cells were Annexin V+/PI+, and necrotic cells were Annexin V−/PI+.

believe that unexplored reasons, not noted here, may be the cause of the difference in cytotoxicity between PE9 and PE10. It could therefore be postulated that Arg4 and Lys6 lower the strength by which alpha-importin binds to PE5 and/or increase the binding by RI. Indeed, in the NMR structure of wild-type HP-RNase, Lys1, which belongs to the NLS of PE5, has a high mobility that is most likely due to the presence of a fixed cluster of positive charges (Arg4, Lys6, and Lys7) in its neighborhood.37 PE5 is an attractive antitumor RNase not only because it is of human origin but also because, compared to other RNases like onconase or RI evading RNases, it exerts its cytotoxic activity on a different cell compartment.14 The cytotoxicity of PE5 is based on its routing into the nucleus which offers to this RNase new RNA substrates and therefore new targets to kill tumor cells. In this sense, we have already shown18 that the mechanism of cytotoxicity of PE5 is different from that of onconase. PE5 is recognized by the alpha-importin17 and by the RI,15 and the regions of the protein implicated in the binding to both proteins overlap. This means that PE5 can bind to each protein but not to both at the same time. The concentration of alphaimportin in the cytosol of Xenopus laevis oocytes has been described to be of 3 μM38 and that of RI in the cytosol of human cell is of 4 μM.39 Thus, it is likely that alpha-importin and RI are found at similar concentrations in the cytosol. In this case, the affinity of the RNase for both proteins would determine to which one it will mainly bind. It has been described that the strength of the binding between HP-RNase and RI has one of the lowest described Kd (2.9 × 10−16 M),40 but several facts may explain how the PE5 molecules that reach the cytosol are driven into the nucleus. First of all, we cannot exclude that the strength of binding of alpha-importin to PE5 could be very high. The fact that PE5 is more cytotoxic than NLSPE3 although this latter variant is more basic than the former one could indicate that the NLS of PE5 is much better recognized by the alpha-importin than that of the SV40 stretch. Second, although the affinity of PE5 by the RI would be higher than that for the alpha-importin, those PE5 molecules captured by the alpha-importin would be released into the nucleus and removed from the two competing equilibriums. The lack of free PE5 molecules would shift the PE5−RI equilibrium toward dissociation rendering free PE5 molecules ready to be captured by the alpha-importin. As a result, PE5 will progressively accumulate into the nucleus. PE3 evades the RI, but its cytotoxicity is low and restricted to some cell lines.15 It is also worth mentioning that we have observed that even though the replacements of Arg31 by Glu or Arg33 by Ala in PE5 increase the ability of these variants to evade the RI in vitro (Tubert et al., unpublished results), these mutations abolish the nuclear import of these PE5 variants16 and are poorly cytotoxic compared to PE5.17 These results

not induce necrosis of the cells even after 72 h of incubation. The percentage of cells in early and late apoptosis was equivalent for the two treatments assayed. We also investigated the activation of procaspases-3, -8, and -9 in NCI/ADR-RES cells after incubation with 4.85 μM NLSPE5 or 44.5 μM PE5 (Figure 6A). NLSPE5 induced the activation of initiator procaspases-8 and -9 and executioner procaspase-3 as previously shown for PE5.18 The pattern of procaspase activation did not differ between the two variants. Procaspase activation was evident at 48 h and increased at 72 h of incubation with the RNases. We had previously shown that PE5 arrests the cell cycle of NCI/ADR-RES cells at S- and G2/M-phases and this is accompanied by a 2-fold accumulation of cyclin E and p21WAF1/CIP1 but unchanged levels of cyclin D1.18 Our results indicated that the cytotoxicity of PE5 was mediated by the increase of the expression of p21WAF1/CIP1, which could explain why PE5 reduces the level of P-gp in MDR cell lines.18,19 We were therefore interested in investigating whether the cytotoxic mechanism of NLSPE5 was similar to that of PE5. We studied by Western blot the effect produced by 0.97 μM NLSPE5 and 8.9 μM PE5 on the accumulation of cyclin D1, cyclin E, and p21WAF1/CIP1 in NCI/ADR-RES cells. Figure 6B shows that NLSPE5, as previously described for PE5, induces a 2-fold accumulation of cyclin E and p21WAF1/CIP1 in this cell line, but does not affect the accumulation of cyclin D1. We had previously shown that the decrease of the expression level of P-gp in MDR cell lines induced by PE5 could be functionally confirmed by measuring the accumulation of doxorubicin in MDR cells treated with the RNase.19 We investigated the doxorubicin accumulation in NCI-H460/R cells treated with different concentrations of NLSPE5 and PE5 by FACS analysis (Figure 6C). As expected, after treatment with NLSPE5 for 72 h, doxorubicin accumulation increased in a dose-dependent manner up to 40% with respect to untreated control cells at the highest RNase concentration assayed. The increase in doxorubicin accumulation was analogous in the two treatments assayed.

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DISCUSSION One major concern in the development of recombinant antitumor drugs is the generation of an immunogenic response against the drug in treated patients. When we tried to revert some of the mutations introduced in PE5 in order to create a new variant with as many residues as possible from the wildtype human sequence, we were surprised that PE9 was much less cytotoxic than PE5 or PE10. Protein stability can be an important factor in the cytotoxic potency of an RNase.33 Although PE10 is 4 °C more thermally stable than PE9, this effect could be counteracted because PE10 lacks two basic residues in comparison to PE9 and, as described, the increase of basicity raises the cytotoxicity of different RNases.34−36 We 2899



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Figure 6. Effects induced by NLSPE5 and PE5 on cancer cells. (A) Procaspase-3, -8, and -9 activation in NCI/ADR-RES cells treated with NLSPE5 or PE5 for 24 h (black bars), 48 h (striped bars), and 72 h (checkered bars). White bars indicate untreated cells (control). (B) Effects on the expression of cyclin D1, cyclin E, and p21WAF1/CIP1 in NCI/ADR-RES cells. Top: Western blot of a representative experiment. Bottom: Densitometric analysis of the GAPDH-normalized immunoblots of each treatment respective to that of untreated cells. White bars indicate untreated cells (control), and black and checkered bars indicate NLSPE5- and PE5-treated cells, respectively. (C) Effect on doxorubicin accumulation in NCIH460/R cells. Cells were treated for 72 h with concentrations of the RNases that produced a decrease of cell proliferation of 20% (striped bars), 40% (black bars), and 50% (checkered bars) and then exposed to 10 μM doxorubicin for 1 h. 10 μM verapamil (white bar) was used as a positive control.

PE5 to reach the nucleus, it could be possible to increase its affinity for the alpha-importin. One strategy to improve the binding of a molecule to a partner is the insertion of additional new binding sites onto the molecule. Natural and synthetic multivalent ligands can bind to receptors with high avidity and specificity and therefore can function as potent effectors or inhibitors of biological processes.41 To this end, we have fused a

indicate that nuclear targeting confers to PE5 more cytotoxic capacity than a potential RI evasion would do. We were aware that the effect of PE5 on cell cultures was lower than that of onconase.18 We were therefore interested in improving the cytotoxicity of PE5 and reasoned that increasing the capacity of the RNase to reach the nucleus would lead to an increase of its cytotoxicity. In order to increase the capacity of 2900



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known and efficient NLS at each end of the protein to increase the binding of PE5 for the alpha-importin. Among the different constructions assayed, only NLSPE5, carrying the NLS at its Nterminus, is clearly more cytotoxic than PE5. Insertion of the same NLS at different loops of the protein was also investigated, but the resulting variants were not cytotoxic, probably because they produced a very unstable protein (results not shown). This indicates that the position where the NLS is inserted into the RNase is critical for its cytotoxic activity. Insertion of the same NLS at the N-terminus of PE3 also produces a clear increase of cytotoxicity indicating that the increase of cytotoxicity promoted by the insertion of the Nterminal NLS is not restricted to PE5. The difference in cytotoxicity between NLSPE5 and scNLSPE5 (Table 2) or between NLSPE3 and scNLSPE3 indicates that the cytotoxicity of NLSPE5 is mainly due to its routing into the nucleus. We are aware that the increase of cytotoxicity observed for NLSPE5 respective to PE5 could be also due in part to the cationization of the protein since the scNLSPE5 is also slightly more cytotoxic than PE5. Cationization of an RNase by chemical or genetic modification has been previously described as a strategy to increase its internalization efficiency.34−36 This strategy is based on the rationale that a more efficient internalization could be achieved by using electrostatic interactions to adsorb highly cationic proteins to the negatively charged membrane surface. Nevertheless, we have shown that the additional basic stretch does not confer a cytotoxic activity per se since the NLSPE5H119A variant, which carries the same stretch and does not have catalytic activity, is not cytotoxic. It is interesting to note that in vitro the binding between NLSPE5 and RI seems slightly lower than for the rest of the variants (Figure 2). However, NLSPE5 is not evading the RI in vivo, since we have observed that it does not cleave cytosolic RNA, an indication that RI is protecting it (Figure 5). Nuclear accumulation determines the cytotoxic properties of the protein. We have obtained multiple data that indicate that NLSPE5 and PE5 share the same cytotoxic mechanism. Treatment with PE5 or NLSPE5 increases 2-fold the accumulation of p21WAF1/CIP1 and cyclin E, but not that of cyclin D1 (Figure 6B), produces a similar accumulation of doxorubicin inside MDR cell lines (Figure 6C) and induces the same proportion of early and late apoptotic cells along the different incubation times (Table 3). Also, the activation pattern of procaspase-3, -8, and -9 is very similar in cells treated with PE5 and NLSPE5 (Figure 6A). All together, the results presented herein indicate that NLSPE5 is an improved form of the nuclear cytotoxic PE5, not because it exerts its cytotoxicity by a different and more effective pathway, but because it cleaves nuclear RNA more efficiently. We envisage the construction of new RNases in which a combination of the two strategies presented here will produce more effective and nonimmunogenic cytotoxic drugs. Furthermore, since NLS-RNases increase the accumulation of other antitumor drugs such as doxorubicin inside MDR cells, we believe that the chemotherapeutic efficacy of NLS-RNases would likely increase as a component of a combination therapy regime. These engineered human RNases are a new generation of non-RI-evading RNases that exert their cytotoxic action by degrading nuclear RNA and are promising agents for the treatment of cancer.

Article

AUTHOR INFORMATION

Corresponding Author

*A.B.: Universitat de Girona, Departament de Biologia, Facultat de Ciències, Campus de Montilivi, M. Aurélia Campmany, 69 17071 Girona, Spain; e-mail, antoni.benito@ udg.edu; fax, +34-972418150. M.V.: e-mail, maria.vilanova@ udg.edu; tel, +34-972418173; fax, +34-972418150. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by Grant BFU2009-06935 from MICINN (Spain) and by Grant GRCT04 from the University of Girona. We are very grateful to Dr. Milica Pesic and Dr. Sabera Ruzdijić for providing us with the NCI-H460/R cell line and to Dr. Ramon Colomer for providing us with the NCI/ ADR-RES cell line. A.V., J.C., and P.T. acknowledge their fellowship from MINEDU, Universitat de Girona, and MEC (Spain), respectively.

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REFERENCES

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