Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1292–1297
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Spectroscopic investigation on the interaction of ruthenium complexes with tumor specific lectin, jacalin Khan Behlol Ayaz Ahmed a, Elamvazhuthi Reshma a, Mariappan Mariappan b, Veerappan Anbazhagan a,⇑ a b
Department of Chemistry, School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudaram, Thanjavur, Tamil Nadu, India Department of Chemistry, SRM University, Kattankulathur, Chennai, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Ruthenium complexes interacts with
Haemagglutination assay: (A) PBS buffer, (B) jacalin, (C) galactose, (D) jacalin–galactose complex, (E) [Ru(phen)3]Cl2, (F) jacalin–[Ru(phen)3]Cl2 complex and (G) jacalin–galactose–[Ru(phen)3]Cl2 complex.
jacalin as good as carbohydrate. Each subunits of the tetrameric jacalin binds one ruthenium complex. Binding sites for the carbohydrate and the ruthenium complexes are different.
A
B
C
D
E
F
G
I II II
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 6 September 2014 Accepted 18 September 2014 Available online 28 September 2014 Keywords: Jacalin Lectin Polypyridyl ruthenium (II) Fluorescence quenching Binding constant
a b s t r a c t Several ruthenium complexes are regarded as anticancer agents and considered as an alternative to the widely used platinum complexes. Owing to the preferential interaction of jacalin with tumor-associated T-antigen, we report the interaction of jacalin with four ruthenium complex namely, tris(1, 10-phenanthroline)ruthenium(II)chloride, bis(1,10-phenanthroline)(N-[1,10]phenanthrolin-5-yl-pyrenylmethanimine)ruthenium(II)chloride, bis(1,10-phenanthroline)(dipyrido[3,2-a:20 ,30 -c]-phenazine) ruthenium(II)chloride, bis(1,10-phenanthroline)(11-(9-acridinyl)dipyrido[3,2-a:20 ,30 -c]phenazine)ruthenium(II) chloride. Fluorescence spectroscopic analysis revealed that the ruthenium complexes strongly quenched the intrinsic fluorescence of jacalin through a static quenching procedure, and a non-radiative energy transfer occurred within the molecules. Association constants obtained for the interaction of different ruthenium complexes with jacalin are in the order of 105 M1, which is in the same range as those obtained for the interaction of lectin with carbohydrate and hydrophobic ligand. Each subunit of the tetrameric jacalin binds one ruthenium complex, and the stoichiometry is found to be unaffected by the presence of the specific sugar, galactose. In addition, agglutination activity of jacalin is largely unaffected by the presence of the ruthenium complexes, indicating that the binding sites for the carbohydrate and the ruthenium complexes are different. These results suggest that the development of lectin–ruthenium complex conjugate would be feasible to target malignant cells in chemo-therapeutics. Ó 2014 Elsevier B.V. All rights reserved.
⇑ Corresponding author. Tel.: +91 4362 264101 3689; fax: +91 4362 264120. E-mail address: [email protected] (V. Anbazhagan). http://dx.doi.org/10.1016/j.saa.2014.09.047 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
K.B. Ayaz Ahmed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1292–1297
Introduction Since the discovery of cisplatin, several platinum based complexes have been synthesized and tested for anticancer activity [1]. Nevertheless, in view of the associated high toxicity of platinum based metal complexes in chemotherapeutics researchers have been searching for new metal complexes with good anticancer property [2]. Among many metal complexes, ruthenium complexes bearing a variety of organo ligands are emerging as a potential anticancer agent. Notably, NAMI-A ([ImH]trans-[RuCl4 (dmso-S)(Im)], Im = Imidazole) and KP1019 ([IndH]trans-[RuCl4 (Ind)2], Ind = Indazole) have been effective against metastasis and have been recommended for phase II clinical trials [3]. Despite the development in metal based chemotherapeutic agents, the exact mechanism of their action remains enigmatic. However, it has been strongly believed that these anticancer agents exert their biological activities through forming adducts with various targets, including DNA, enzymes and metallo-proteases. These adducts can activate DNA damage pathways or inhibit the enzymatic activity [4]. Because of this, the metal-based chemotherapeutic agents are recognized as highly toxic not only to the malignant cells but also to the normal cells [5]. In order to minimize its toxicity to normal cells and to increase their therapeutic potential, it is imperative to increase its bioavailability more specifically in the target tissues. A possible approach is to conjugate the ruthenium complexes to another agent that can steer it to the tumor tissue. Proteins are good candidate to conjugate drug and have emerged as a versatile carriers for the diagnosis and treatment of many diseases, including cancer, diabetes and rheumatoid arthritis [6]. Ruthenium complexes are known to interact with proteins like, bovine serum albumin, apotransferrin, human lactoferrin and apolactoferrin [7], but there are no reports on their interaction with lectins. Lectins are carbohydrate binding proteins of non-immune origin and have been regarded as effective drug carriers [8]. For example, Concanavalin A (ConA) has been used as an effective carrier for the anti-cancer drug, daunomycin [9]. Lectins exert their biological effects in cell–cell recognition, host pathogen interactions, cellular signaling and differentiation and immune responses through binding to appropriate carbohydrates [10]. Several lectins are known for their ability to distinguish between normal and malignant cells and to specifically recognize different types of human blood groups [11]. In this study, we choose Artocarpus integrifolia (jack fruit) lectin (generally called as jacalin), because it specifically recognizes the tumor-associated T-antigenic disaccharide, 2-acetamido,2-deoxy, 3-O-b-D-galctopyranosyl-aD-galactopyranoside (Galb13GalNAca) [12]. Jacalin also has the ability to inhibit HIV infection [13]. Due to its ability to specifically stimulate CD4 cells in comparison to primary T cells, jacalin has been utilized in the AIDS research [14]. Jacalin is a homotetrameric protein of Mr 66 kDa with high specificity for galactose [15]. Fluorescence quenching studies have suggested that the tryptophan residues of jacalin are deeply buried in the hydrophobic core of the protein matrix [16]. Jacalin preferentially binds sugar derivatives bearing a hydrophobic moiety, such as 4-methylumbelliferyl glycosides with greater affinity than simple methyl glycosides of galactose, suggesting that there might be a hydrophobic site in the vicinity of the saccharide-binding site [17]. The carbohydrate specificity of this lectin and the mechanism of interaction have already been reported in detail [18a, 12, 18c–e]. Other than carbohydrates, jacalin also binds to porphyrins, phycocyanin and gold nanoparticles with considerable affinity and the interaction is mediated by hydrophobic forces [19]. The three dimensional structure of jacalin complexed with methyla-D-galactopyranoside and porphyrin have also been solved by single-crystal X-ray diffraction studies [12,20]. These studies suggested that the carbohydrate binding site of jacalin exhibits
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structural plasticity to recognize other hydrophobic molecules. Therefore, it would be possible to prepare jacalin-drug conjugate vehicle for target delivery of ruthenium complexes to the malignant tissues. As the first step in this direction, we have investigated the interaction of jacalin with polypyridyl ruthenium(II) system, namely tris(1,10-phenanthroline)ruthenium(II)chloride [Ru(phen)3]Cl2 [21], bis(1,10-phenanthroline)(N[1,10]phenanthrolin-5-yl-pyrenylmethanimine)ruthenium(II)chloride [Ru(phen)2ppym]Cl2, bis(1,10-phenanthroline)(dipyrido [3,2-a:20 ,30 -c]-phenazine) ruthenium(II)chloride [Ru(phen)2dppz]Cl2 [22], bis(1, 10-phenanthroline)(11-(9-acridinyl)dipyrido[3,2-a:20 ,30 -c]phenazine)ruthenium(II)chloride [Ru(phen)2acdppz]Cl2 [23]. Polypyridyl ruthenium (II) classes of metallointercalators have received particular attention, due to their luminescent behavior and strong affinity to DNA. One important characteristic of these ligands is that they all possess extended p-aromatic structures and are planar, which is crucial for the intercalation of the Ru(II) complexes [24]. Previous reports show that these ruthenium complexes intercalate DNA molecule and induce DNA photo cleavage through the generation of reactive oxygen species [23,24]. Materials and methods Materials The A. integrifolia (Jack fruit) seeds were obtained from local seed vendors. Guar gum was obtained from Loba, India. Sodium phosphate dibasic and monobasic, sodium chloride, epichlorohydrin, sodium citrate, citric acid and galactose were purchased from Merck. Acrylamide, bis-acrylamide and sodium dodecyl sulfate were purchased from Sigma, India. All other reagents were of analytical grade. Preparation of ruthenium complexes [Ru(phen)3]Cl2 [21], [Ru(phen)2dppz]Cl2 [22] and [Ru(phen)2acdppz]Cl2 [23] were prepared as described previously. [Ru(phen)2ppym]Cl2 is newly designed and the complete characterization of [Ru(phen)2ppym]Cl2 and the ligand ppym will be published elsewhere. Briefly, ppym was prepared by Schiff’s base condensation of 5-amino-phenanthroline and 1-pyrenecarboxaldehyde using glacial acetic acid as a catalyst. Its ruthenium complex was prepared by refluxing ppym with the [Ru(phen)2Cl2] 2H2O [25] precursor and precipitated using a saturated solution of NH4PF6. The hexafluorophosphate salts of the complexes were converted into the water-soluble chloride complexes by treating the former salt solutions with an excess of TBACl in acetone. The chloride salts, which are insoluble in acetone, instantaneously precipitate. They were filtered and vacuum-dried before use. The yield was about 90% of the theoretical value in each case. Purification of jacalin Jacalin was purified by affinity chromatography on cross-linked guar gum as described previously [17]. The purity of the protein was assessed by polyacrylamide gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate. The concentration of jacalin was determined by Lowry assay using bovine serum albumin as the standard [26]. Binding of ruthenium complexes to jacalin Fluorescence spectra were recorded on a Jasco-FP8200 spectrofluorimeter. The spectral slit width was set to 2.5 nm for both excitation and emission monochromators. The intrinsic fluorescence
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spectra of jacalin were recorded at 300–400 nm at an excitation wavelength of 280 nm. A fixed volume of jacalin solution (3.0 ml, 7.5 lM) was titrated by adding small aliquots of the Ru-complex from a concentrated stock solution and the fluorescence intensity was recorded after an equilibration period of 2 min. All binding experiments were performed in 10 mM phosphate buffer, containing 0.15 M NaCl, pH 7.4 (PBS). All titrations were repeated at least three times to arrive at average values. Fluorescence intensities were corrected for volume changes and for inner filter effects before further analysis. Lectin activity assay The activity of the protein was checked by haemagglutination and haemagglutination inhibition assays as described in Ref. [27]. In order determine whether ruthenium complex binding altered the sugar-binding activity of the lectin, the haemagglutination experiments were conducted by incubating jacalin with high concentration of ruthenium complex as used in the fluorescence studies. Result and discussion Jacalin exhibits high specificity to Galb13GalNAca, a chemically well-defined tumor associated antigen overexpressed in more than 85% of human carcinomas, including colon, prostate, bladder, cavity and breast [12]. Owing to the importance of ruthenium complex in chemotherapeutics and the preferential interaction of lectin with tumor cells, this study were initiated to investigate the interaction of various ruthenium complexes with jacalin in the presence and absence of the specific sugar, galactose. The structures of the ruthenium complex investigated in this study are shown in Fig. 1. Fluorescence spectra of jacalin in the absence as well as in the presence of different concentration of [Ru(phen)3]Cl2, [Ru(phen)2ppym]Cl2, [Ru(phen)2dppz]Cl2 and [Ru(phen)2acdppz]Cl2 are given in Fig. 2. When excited at 280 nm, jacalin shows an emission at 330 nm suggesting that the protein fluorophores are deeply buried
in the hydrophobic core of the lectin. Addition of the drugs neither changed the shape nor the maximum of jacalin fluorescence emission spectra, indicating that under the given experimental conditions no conformational changes are involved in drug binding to jacalin. Furthermore, ruthenium complex binding to jacalin does not affect the far-UV circular dichorism spectra, indicating no substantial changes occur in the protein secondary structure upon drug binding (data not shown). However, the fluorescence emission intensity (mainly originating form tryptophan and tyrosine residues) at 330 nm gradually decreased after addition of successive aliquots of the drug (Fig. 2). At 10 lM concentration of the drug, [Ru(phen)3]Cl2, [Ru(phen)2ppym]Cl2, [Ru(phen)2dppz]Cl2 and [Ru(phen)2acdppz]Cl2 quenched the fluorescence intensity of jacalin by 51.07%, 51.95%, 70.31% and 62.07%, respectively. The observed fluorescence quenching probably arises from energy transfer occurring between jacalin and ruthenium complexes. The fluorescence quenching mechanism can be analyzed with the Stern–Volmer equation [28].
F 0 =F c ¼ 1 þ K sv ½Q ¼ 1 þ K q s0 ½Q
ð1Þ
K q ¼ K sv =s0
ð2Þ
where F0 and Fc are the relative fluorescence intensities of jacalin at 330 nm in the absence and presence of drug, respectively, Ksv is the Stern–Volmer fluorescence quenching constant, [Q] is the concentration of the drug, Kq is the biomolecular quenching constant and s0 is the average fluorescence lifetime (109 s for lectin) [28]. Fig. 3 shows the typical Stern–Volmer plots for jacalin–ruthenium complexes, which display good linear relationships with increasing concentration of the drug. The quenching constants for jacalin in the presence of drug were all close to 1013 L/mol s. For any diffusion-controlled quenching process, the maximum value of Kq is 1010 L/mol s [28]. Therefore, the higher values of Kq obtained in this study indicates the quenching in jacalin-drug systems is a static rather than a dynamic quenching mechanism. Thus, a non-fluorescent fluorophore–drug complex is likely to be formed between the ruthenium complex and jacalin. Representative binding curve for the association of [Ru(phen)3]Cl2 with jacalin are shown in Fig. 4. The binding curves show that
2+ N N
N Ru N
2+
N
N
N
N
N Ru N
N N
A
N
B 2+
N N
N Ru N
C
2+
N
N
N
N
N
N
N Ru N
N
N
N
N
N
D
Fig. 1. Structure of the ruthenium complexes used in this study. (A) tris(1,10-phenanthroline) ruthenium(II)chloride [Ru(phen)3]Cl2; (B) bis(1,10-phenanthroline)(N-[1,10] phenanthrolin-5-yl-pyrenylmethanimine) ruthenium(II)chloride [Ru(phen)2ppym]Cl2; (C) bis(1,10-phenanthroline)(dipyrido[3,2-a:20 ,30 -c]-phenazine) ruthenium(II)chloride [Ru(phen)2dppz]Cl2; (D) bis(1,10-phenanthroline)(11-(9-acridinyl)dipyrido[3,2-a:20 ,30 -c]phenazine)ruthenium(II)chloride [Ru(phen)2acdppz]Cl2.
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Fig. 2. Jacalin fluorescence emission spectra monitored after addition of increasing concentrations of (A) [Ru(phen)3]Cl2, (B) [Ru(phen)2ppym]Cl2 (C) [Ru(phen)2dppz]Cl2 and (D) [Ru(phen)2acdppz]Cl2. The upper spectrum in each panel corresponds to free jacalin and the remaining spectra with decreasing fluorescence emission were obtained in the presence of increasing concentrations of ruthenium complex.
Fig. 3. Stern–Volmer plot for jacalin binding of (j) [Ru(phen)3]Cl2, (h) [Ru(phen)2ppym]Cl2 (d) [Ru(phen)2dppz]Cl2 and (s) [Ru(phen)2acdppz]Cl2.
the change in fluorescence intensity (DF) increases with increasing drug concentration initially, but displays saturation behavior at very high concentrations of the drug. From this binding data, the association constant (Ka) and the number of binding sites (n) between the protein and drug can be calculated by the following equation [29]
logðF 0 F c =F c F 1 Þ ¼ log K a þ n log½Q
ð3Þ
where F1 is the change in fluorescence intensity at infinite drug concentration, n is the number of binding site and Ka is the
Fig. 4. Representative binding curve for the interaction of [Ru(phen)3]Cl2 with jacalin. The change in fluorescence at 330 nm resulting from the addition of drug to the lectin is plotted as function of the total drug concentration. Inset: plot of F0/DF as function of the reciprocal total drug concentration. The reciprocal of the Yintercept of this plot gave the value of DF1, the change in fluorescence intensity, when all the protein molecules are bound by the drug.
association constant. F1 was obtained from the ordinate intercept of the plot of F0/DF vs 1/[Q] (inset of Fig. 4). A double logarithmic plot for the interaction of ruthenium complex with jacalin is given in Fig. 5. Table 1 shows the values of Ka and n of jacalin binding to ruthenium complex. The values of n at the experimental temperatures are all nearly close to 1, indicating that each lectin subunit binds one ruthenium complex. The observed differences in the binding constant for different ruthenium complex indicates the interaction between the lectin and metal complex depends on the structure of the coordinating ligand.
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A
B
C
D
E
F
G
I II II
Fig. 5. Analysis of the fluorescence titration with (j) [Ru(phen)3]Cl2, (h) [Ru(phen)2ppym]Cl2 (d) [Ru(phen)2dppz]Cl2 and (s) [Ru(phen)2acdppz]Cl2. A plot of log [(F0–Fc/Fc–F1)] against log [drug] was analyzed according to [30].
In order to test the effect of specific sugar on the ruthenium complex binding characteristics of jacalin, we determined the association constant in the presence of 0.1 M galactose. As can be seen from Table 1, the Ka values obtained in the absence and in the presence of galactose differ somewhat. However, the stoichiometry of binding remains unchanged indicating that galactose binding does not inhibit the interaction of ruthenium complex. Agglutinationinhibition assay indicated no loss of erythrocyte-agglutination activity by jacalin in the presence of ruthenium complex (Fig. 6, column F). However, addition of 0.1 M galactose to jacalin–ruthenium complex inhibits agglutination indicating that the binding sites on jacalin for the ruthenium complex and sugar are distinctly different (Fig. 6, column G). These results also suggest that jacalin remains in the native functional state while interacting with the ruthenium complex. The order of Ka for jacalin–ruthenium complex interaction is about 105 M1, which is similar that of lectinsugar binding and lectin interaction with other hydrophobic ligands [19]. Considering the strength of the jacalin–ruthenium complex interactions, it is likely that the maximum percentage of the ruthenium complex is expected to be in the lectin bound state. This is crucial for any therapeutic application, because it could deliver more amount of drug selectively to the target tissues. These data also suggest the possibility of some endogenous hydrophobic ligands exist for jacalin in their native tissues. Recently, jacalin-phthalocyanine-gold nanopartilces conjugate were used to target HT-29 human colorectal adenocarcinoma cells [30]. Previously, ConA-diphtheria toxin conjugate was shown to be effective against CHO and SV3T3 cultured cells and animal models [31]. ConA-daunomycin conjugate have been successfully tested on tumor-bearing mice [9]. Preliminary studies showed that the ruthenium complexes used in this study have the potential to inhibit the growth of human breast cancer cell line (ZR-75-30) (unpublished observation). In light of these observations, we propose that jacalin is extremely well suited for the binding of a variety of metal-based complexes under laboratory conditions and has the potential to be useful as vehicle for targeting such complexes to specific tissues in chemotherapeutics.
Fig. 6. Haemagglutination assays with human erythrocytes. (A) PBS buffer, (B) Jacalin, (C) galactose, (D) jacalin–galactose complex, (E) [Ru(phen)3]Cl2, (F) Jacalin– [Ru(phen)3]Cl2 complex and (G) Jacalin–galactose–[Ru(phen)3]Cl2 complex. About 100 lL of 4% human erythrocytes are used in this experiment. Jacalain agglutinates erythrocytes whereas the presence of galactose inhibits the agglutination. As inferred from this data, agglutination was only inhibited by the specific sugar galactose (D) and not by [Ru(phen)3]Cl2 (G), suggesting that [Ru(phen)3]Cl2 and galactose bind at different site. Experiments were performed in triplicate.
Conclusion In summary, this paper reports the first demonstration of the interaction of ruthenium complex with a lectin. Ruthenium complexes investigated here bind to jacalin with significantly high affinities that are comparable to the interaction of monosaccharides to this tumor-specific lectin and the interaction is mediated predominantly by hydrophobic forces. In view of the current interest in ruthenium complexes as anti-cancer agents, we envisage that a judicious use of ruthenium complex–jacalin mixtures or conjugates will be useful in increasing the drug efficacy in chemotherapeutics. Moreover, jack fruit seeds forms part of the diet in the tropics, therefore, it is also possible to administrate ruthenium–lectin complexes through oral route. Currently, studies employing cell cultures and animal models are in progress and the results will be reported in due course. Acknowledgment KBAA earnestly acknowledges the teaching assistantship from SASTRA University. VA acknowledges the financial support through Department of Science and Technology, Government of India (SB/FT/LS-217/2012). The authors thank Central Research Facility, SASTRA University for providing the necessary infrastructure. References [1] I. Ali, W.A. Wani, K. Saleem, A. Haque, Anticancer Agents Med. Chem. 13 (2013) 296–306. [2] N. Muhammad, Z. Guo, Curr. Opin. Chem. Biol. 19C (2014) 144–153. [3] (a) B.M. Blunden, A. Rawal, H. Lu, M.H. Stenzel, Macromolecules 47 (2014) 1646–1655; (b) Rademaker-Lakhai, D. van den Bongard, D. Pluim, J.H. Beijnen, J.H.M. Schellens, Clin. Cancer Res. 10 (2004) 3717–3727; (c) C. Hartinger, S. Zorbas-Seifried, M. Jakupec, B. Kynast, H. Zorbas, B. Keppler, J. Inorg. Biochem. 100 (2006) 891–904; (d) E. Alessio, G. Mestroni, A. Bergamo, G. Sava, Curr. Top. Med. Chem. 4 (2004) 1525–1535.
Table 1 Stern–Volmer quenching constant (Ksv), binding constant (Ka) and number of binding site of (n) of jacalin interacting with different ruthenium complexes. Molecule
Without 0.1 M galactose 4
Ksv 10 [Ru(phen)3]Cl2 [Ru(phen)2ppym]Cl2 [Ru(phen)2dppz]Cl2 [Ru(phen)2acdppz]Cl2
8.24 8.69 19.77 15.89
(M
1
)
With 0.1 M galactose n
Ka 10
0.98 1.06 1.00 1.08
1.86 2.27 7.08 5.62
5
1
(M
)
Ksv 104 (M1)
n
Ka 105 (M1)
9.26 6.59 13.64 12.79
1.09 0.96 0.98 1.12
2.18 3.48 6.59 4.32
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Spectroscopic investigation on the interaction of ruthenium complexes with tumor specific lectin, jacalin Khan Behlol Ayaz Ahmed a, Elamvazhuthi Reshma a, Mariappan Mariappan b, Veerappan Anbazhagan a,⇑ a b
Department of Chemistry, School of Chemical and Biotechnology, SASTRA University, Thirumalaisamudaram, Thanjavur, Tamil Nadu, India Department of Chemistry, SRM University, Kattankulathur, Chennai, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Ruthenium complexes interacts with
Haemagglutination assay: (A) PBS buffer, (B) jacalin, (C) galactose, (D) jacalin–galactose complex, (E) [Ru(phen)3]Cl2, (F) jacalin–[Ru(phen)3]Cl2 complex and (G) jacalin–galactose–[Ru(phen)3]Cl2 complex.
jacalin as good as carbohydrate. Each subunits of the tetrameric jacalin binds one ruthenium complex. Binding sites for the carbohydrate and the ruthenium complexes are different.
A
B
C
D
E
F
G
I II II
a r t i c l e
i n f o
Article history: Received 16 June 2014 Received in revised form 6 September 2014 Accepted 18 September 2014 Available online 28 September 2014 Keywords: Jacalin Lectin Polypyridyl ruthenium (II) Fluorescence quenching Binding constant
a b s t r a c t Several ruthenium complexes are regarded as anticancer agents and considered as an alternative to the widely used platinum complexes. Owing to the preferential interaction of jacalin with tumor-associated T-antigen, we report the interaction of jacalin with four ruthenium complex namely, tris(1, 10-phenanthroline)ruthenium(II)chloride, bis(1,10-phenanthroline)(N-[1,10]phenanthrolin-5-yl-pyrenylmethanimine)ruthenium(II)chloride, bis(1,10-phenanthroline)(dipyrido[3,2-a:20 ,30 -c]-phenazine) ruthenium(II)chloride, bis(1,10-phenanthroline)(11-(9-acridinyl)dipyrido[3,2-a:20 ,30 -c]phenazine)ruthenium(II) chloride. Fluorescence spectroscopic analysis revealed that the ruthenium complexes strongly quenched the intrinsic fluorescence of jacalin through a static quenching procedure, and a non-radiative energy transfer occurred within the molecules. Association constants obtained for the interaction of different ruthenium complexes with jacalin are in the order of 105 M1, which is in the same range as those obtained for the interaction of lectin with carbohydrate and hydrophobic ligand. Each subunit of the tetrameric jacalin binds one ruthenium complex, and the stoichiometry is found to be unaffected by the presence of the specific sugar, galactose. In addition, agglutination activity of jacalin is largely unaffected by the presence of the ruthenium complexes, indicating that the binding sites for the carbohydrate and the ruthenium complexes are different. These results suggest that the development of lectin–ruthenium complex conjugate would be feasible to target malignant cells in chemo-therapeutics. Ó 2014 Elsevier B.V. All rights reserved.
⇑ Corresponding author. Tel.: +91 4362 264101 3689; fax: +91 4362 264120. E-mail address: [email protected] (V. Anbazhagan). http://dx.doi.org/10.1016/j.saa.2014.09.047 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
K.B. Ayaz Ahmed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1292–1297
Introduction Since the discovery of cisplatin, several platinum based complexes have been synthesized and tested for anticancer activity [1]. Nevertheless, in view of the associated high toxicity of platinum based metal complexes in chemotherapeutics researchers have been searching for new metal complexes with good anticancer property [2]. Among many metal complexes, ruthenium complexes bearing a variety of organo ligands are emerging as a potential anticancer agent. Notably, NAMI-A ([ImH]trans-[RuCl4 (dmso-S)(Im)], Im = Imidazole) and KP1019 ([IndH]trans-[RuCl4 (Ind)2], Ind = Indazole) have been effective against metastasis and have been recommended for phase II clinical trials [3]. Despite the development in metal based chemotherapeutic agents, the exact mechanism of their action remains enigmatic. However, it has been strongly believed that these anticancer agents exert their biological activities through forming adducts with various targets, including DNA, enzymes and metallo-proteases. These adducts can activate DNA damage pathways or inhibit the enzymatic activity [4]. Because of this, the metal-based chemotherapeutic agents are recognized as highly toxic not only to the malignant cells but also to the normal cells [5]. In order to minimize its toxicity to normal cells and to increase their therapeutic potential, it is imperative to increase its bioavailability more specifically in the target tissues. A possible approach is to conjugate the ruthenium complexes to another agent that can steer it to the tumor tissue. Proteins are good candidate to conjugate drug and have emerged as a versatile carriers for the diagnosis and treatment of many diseases, including cancer, diabetes and rheumatoid arthritis [6]. Ruthenium complexes are known to interact with proteins like, bovine serum albumin, apotransferrin, human lactoferrin and apolactoferrin [7], but there are no reports on their interaction with lectins. Lectins are carbohydrate binding proteins of non-immune origin and have been regarded as effective drug carriers [8]. For example, Concanavalin A (ConA) has been used as an effective carrier for the anti-cancer drug, daunomycin [9]. Lectins exert their biological effects in cell–cell recognition, host pathogen interactions, cellular signaling and differentiation and immune responses through binding to appropriate carbohydrates [10]. Several lectins are known for their ability to distinguish between normal and malignant cells and to specifically recognize different types of human blood groups [11]. In this study, we choose Artocarpus integrifolia (jack fruit) lectin (generally called as jacalin), because it specifically recognizes the tumor-associated T-antigenic disaccharide, 2-acetamido,2-deoxy, 3-O-b-D-galctopyranosyl-aD-galactopyranoside (Galb13GalNAca) [12]. Jacalin also has the ability to inhibit HIV infection [13]. Due to its ability to specifically stimulate CD4 cells in comparison to primary T cells, jacalin has been utilized in the AIDS research [14]. Jacalin is a homotetrameric protein of Mr 66 kDa with high specificity for galactose [15]. Fluorescence quenching studies have suggested that the tryptophan residues of jacalin are deeply buried in the hydrophobic core of the protein matrix [16]. Jacalin preferentially binds sugar derivatives bearing a hydrophobic moiety, such as 4-methylumbelliferyl glycosides with greater affinity than simple methyl glycosides of galactose, suggesting that there might be a hydrophobic site in the vicinity of the saccharide-binding site [17]. The carbohydrate specificity of this lectin and the mechanism of interaction have already been reported in detail [18a, 12, 18c–e]. Other than carbohydrates, jacalin also binds to porphyrins, phycocyanin and gold nanoparticles with considerable affinity and the interaction is mediated by hydrophobic forces [19]. The three dimensional structure of jacalin complexed with methyla-D-galactopyranoside and porphyrin have also been solved by single-crystal X-ray diffraction studies [12,20]. These studies suggested that the carbohydrate binding site of jacalin exhibits
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structural plasticity to recognize other hydrophobic molecules. Therefore, it would be possible to prepare jacalin-drug conjugate vehicle for target delivery of ruthenium complexes to the malignant tissues. As the first step in this direction, we have investigated the interaction of jacalin with polypyridyl ruthenium(II) system, namely tris(1,10-phenanthroline)ruthenium(II)chloride [Ru(phen)3]Cl2 [21], bis(1,10-phenanthroline)(N[1,10]phenanthrolin-5-yl-pyrenylmethanimine)ruthenium(II)chloride [Ru(phen)2ppym]Cl2, bis(1,10-phenanthroline)(dipyrido [3,2-a:20 ,30 -c]-phenazine) ruthenium(II)chloride [Ru(phen)2dppz]Cl2 [22], bis(1, 10-phenanthroline)(11-(9-acridinyl)dipyrido[3,2-a:20 ,30 -c]phenazine)ruthenium(II)chloride [Ru(phen)2acdppz]Cl2 [23]. Polypyridyl ruthenium (II) classes of metallointercalators have received particular attention, due to their luminescent behavior and strong affinity to DNA. One important characteristic of these ligands is that they all possess extended p-aromatic structures and are planar, which is crucial for the intercalation of the Ru(II) complexes [24]. Previous reports show that these ruthenium complexes intercalate DNA molecule and induce DNA photo cleavage through the generation of reactive oxygen species [23,24]. Materials and methods Materials The A. integrifolia (Jack fruit) seeds were obtained from local seed vendors. Guar gum was obtained from Loba, India. Sodium phosphate dibasic and monobasic, sodium chloride, epichlorohydrin, sodium citrate, citric acid and galactose were purchased from Merck. Acrylamide, bis-acrylamide and sodium dodecyl sulfate were purchased from Sigma, India. All other reagents were of analytical grade. Preparation of ruthenium complexes [Ru(phen)3]Cl2 [21], [Ru(phen)2dppz]Cl2 [22] and [Ru(phen)2acdppz]Cl2 [23] were prepared as described previously. [Ru(phen)2ppym]Cl2 is newly designed and the complete characterization of [Ru(phen)2ppym]Cl2 and the ligand ppym will be published elsewhere. Briefly, ppym was prepared by Schiff’s base condensation of 5-amino-phenanthroline and 1-pyrenecarboxaldehyde using glacial acetic acid as a catalyst. Its ruthenium complex was prepared by refluxing ppym with the [Ru(phen)2Cl2] 2H2O [25] precursor and precipitated using a saturated solution of NH4PF6. The hexafluorophosphate salts of the complexes were converted into the water-soluble chloride complexes by treating the former salt solutions with an excess of TBACl in acetone. The chloride salts, which are insoluble in acetone, instantaneously precipitate. They were filtered and vacuum-dried before use. The yield was about 90% of the theoretical value in each case. Purification of jacalin Jacalin was purified by affinity chromatography on cross-linked guar gum as described previously [17]. The purity of the protein was assessed by polyacrylamide gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate. The concentration of jacalin was determined by Lowry assay using bovine serum albumin as the standard [26]. Binding of ruthenium complexes to jacalin Fluorescence spectra were recorded on a Jasco-FP8200 spectrofluorimeter. The spectral slit width was set to 2.5 nm for both excitation and emission monochromators. The intrinsic fluorescence
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spectra of jacalin were recorded at 300–400 nm at an excitation wavelength of 280 nm. A fixed volume of jacalin solution (3.0 ml, 7.5 lM) was titrated by adding small aliquots of the Ru-complex from a concentrated stock solution and the fluorescence intensity was recorded after an equilibration period of 2 min. All binding experiments were performed in 10 mM phosphate buffer, containing 0.15 M NaCl, pH 7.4 (PBS). All titrations were repeated at least three times to arrive at average values. Fluorescence intensities were corrected for volume changes and for inner filter effects before further analysis. Lectin activity assay The activity of the protein was checked by haemagglutination and haemagglutination inhibition assays as described in Ref. [27]. In order determine whether ruthenium complex binding altered the sugar-binding activity of the lectin, the haemagglutination experiments were conducted by incubating jacalin with high concentration of ruthenium complex as used in the fluorescence studies. Result and discussion Jacalin exhibits high specificity to Galb13GalNAca, a chemically well-defined tumor associated antigen overexpressed in more than 85% of human carcinomas, including colon, prostate, bladder, cavity and breast [12]. Owing to the importance of ruthenium complex in chemotherapeutics and the preferential interaction of lectin with tumor cells, this study were initiated to investigate the interaction of various ruthenium complexes with jacalin in the presence and absence of the specific sugar, galactose. The structures of the ruthenium complex investigated in this study are shown in Fig. 1. Fluorescence spectra of jacalin in the absence as well as in the presence of different concentration of [Ru(phen)3]Cl2, [Ru(phen)2ppym]Cl2, [Ru(phen)2dppz]Cl2 and [Ru(phen)2acdppz]Cl2 are given in Fig. 2. When excited at 280 nm, jacalin shows an emission at 330 nm suggesting that the protein fluorophores are deeply buried
in the hydrophobic core of the lectin. Addition of the drugs neither changed the shape nor the maximum of jacalin fluorescence emission spectra, indicating that under the given experimental conditions no conformational changes are involved in drug binding to jacalin. Furthermore, ruthenium complex binding to jacalin does not affect the far-UV circular dichorism spectra, indicating no substantial changes occur in the protein secondary structure upon drug binding (data not shown). However, the fluorescence emission intensity (mainly originating form tryptophan and tyrosine residues) at 330 nm gradually decreased after addition of successive aliquots of the drug (Fig. 2). At 10 lM concentration of the drug, [Ru(phen)3]Cl2, [Ru(phen)2ppym]Cl2, [Ru(phen)2dppz]Cl2 and [Ru(phen)2acdppz]Cl2 quenched the fluorescence intensity of jacalin by 51.07%, 51.95%, 70.31% and 62.07%, respectively. The observed fluorescence quenching probably arises from energy transfer occurring between jacalin and ruthenium complexes. The fluorescence quenching mechanism can be analyzed with the Stern–Volmer equation [28].
F 0 =F c ¼ 1 þ K sv ½Q ¼ 1 þ K q s0 ½Q
ð1Þ
K q ¼ K sv =s0
ð2Þ
where F0 and Fc are the relative fluorescence intensities of jacalin at 330 nm in the absence and presence of drug, respectively, Ksv is the Stern–Volmer fluorescence quenching constant, [Q] is the concentration of the drug, Kq is the biomolecular quenching constant and s0 is the average fluorescence lifetime (109 s for lectin) [28]. Fig. 3 shows the typical Stern–Volmer plots for jacalin–ruthenium complexes, which display good linear relationships with increasing concentration of the drug. The quenching constants for jacalin in the presence of drug were all close to 1013 L/mol s. For any diffusion-controlled quenching process, the maximum value of Kq is 1010 L/mol s [28]. Therefore, the higher values of Kq obtained in this study indicates the quenching in jacalin-drug systems is a static rather than a dynamic quenching mechanism. Thus, a non-fluorescent fluorophore–drug complex is likely to be formed between the ruthenium complex and jacalin. Representative binding curve for the association of [Ru(phen)3]Cl2 with jacalin are shown in Fig. 4. The binding curves show that
2+ N N
N Ru N
2+
N
N
N
N
N Ru N
N N
A
N
B 2+
N N
N Ru N
C
2+
N
N
N
N
N
N
N Ru N
N
N
N
N
N
D
Fig. 1. Structure of the ruthenium complexes used in this study. (A) tris(1,10-phenanthroline) ruthenium(II)chloride [Ru(phen)3]Cl2; (B) bis(1,10-phenanthroline)(N-[1,10] phenanthrolin-5-yl-pyrenylmethanimine) ruthenium(II)chloride [Ru(phen)2ppym]Cl2; (C) bis(1,10-phenanthroline)(dipyrido[3,2-a:20 ,30 -c]-phenazine) ruthenium(II)chloride [Ru(phen)2dppz]Cl2; (D) bis(1,10-phenanthroline)(11-(9-acridinyl)dipyrido[3,2-a:20 ,30 -c]phenazine)ruthenium(II)chloride [Ru(phen)2acdppz]Cl2.
K.B. Ayaz Ahmed et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1292–1297
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Fig. 2. Jacalin fluorescence emission spectra monitored after addition of increasing concentrations of (A) [Ru(phen)3]Cl2, (B) [Ru(phen)2ppym]Cl2 (C) [Ru(phen)2dppz]Cl2 and (D) [Ru(phen)2acdppz]Cl2. The upper spectrum in each panel corresponds to free jacalin and the remaining spectra with decreasing fluorescence emission were obtained in the presence of increasing concentrations of ruthenium complex.
Fig. 3. Stern–Volmer plot for jacalin binding of (j) [Ru(phen)3]Cl2, (h) [Ru(phen)2ppym]Cl2 (d) [Ru(phen)2dppz]Cl2 and (s) [Ru(phen)2acdppz]Cl2.
the change in fluorescence intensity (DF) increases with increasing drug concentration initially, but displays saturation behavior at very high concentrations of the drug. From this binding data, the association constant (Ka) and the number of binding sites (n) between the protein and drug can be calculated by the following equation [29]
logðF 0 F c =F c F 1 Þ ¼ log K a þ n log½Q
ð3Þ
where F1 is the change in fluorescence intensity at infinite drug concentration, n is the number of binding site and Ka is the
Fig. 4. Representative binding curve for the interaction of [Ru(phen)3]Cl2 with jacalin. The change in fluorescence at 330 nm resulting from the addition of drug to the lectin is plotted as function of the total drug concentration. Inset: plot of F0/DF as function of the reciprocal total drug concentration. The reciprocal of the Yintercept of this plot gave the value of DF1, the change in fluorescence intensity, when all the protein molecules are bound by the drug.
association constant. F1 was obtained from the ordinate intercept of the plot of F0/DF vs 1/[Q] (inset of Fig. 4). A double logarithmic plot for the interaction of ruthenium complex with jacalin is given in Fig. 5. Table 1 shows the values of Ka and n of jacalin binding to ruthenium complex. The values of n at the experimental temperatures are all nearly close to 1, indicating that each lectin subunit binds one ruthenium complex. The observed differences in the binding constant for different ruthenium complex indicates the interaction between the lectin and metal complex depends on the structure of the coordinating ligand.
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A
B
C
D
E
F
G
I II II
Fig. 5. Analysis of the fluorescence titration with (j) [Ru(phen)3]Cl2, (h) [Ru(phen)2ppym]Cl2 (d) [Ru(phen)2dppz]Cl2 and (s) [Ru(phen)2acdppz]Cl2. A plot of log [(F0–Fc/Fc–F1)] against log [drug] was analyzed according to [30].
In order to test the effect of specific sugar on the ruthenium complex binding characteristics of jacalin, we determined the association constant in the presence of 0.1 M galactose. As can be seen from Table 1, the Ka values obtained in the absence and in the presence of galactose differ somewhat. However, the stoichiometry of binding remains unchanged indicating that galactose binding does not inhibit the interaction of ruthenium complex. Agglutinationinhibition assay indicated no loss of erythrocyte-agglutination activity by jacalin in the presence of ruthenium complex (Fig. 6, column F). However, addition of 0.1 M galactose to jacalin–ruthenium complex inhibits agglutination indicating that the binding sites on jacalin for the ruthenium complex and sugar are distinctly different (Fig. 6, column G). These results also suggest that jacalin remains in the native functional state while interacting with the ruthenium complex. The order of Ka for jacalin–ruthenium complex interaction is about 105 M1, which is similar that of lectinsugar binding and lectin interaction with other hydrophobic ligands [19]. Considering the strength of the jacalin–ruthenium complex interactions, it is likely that the maximum percentage of the ruthenium complex is expected to be in the lectin bound state. This is crucial for any therapeutic application, because it could deliver more amount of drug selectively to the target tissues. These data also suggest the possibility of some endogenous hydrophobic ligands exist for jacalin in their native tissues. Recently, jacalin-phthalocyanine-gold nanopartilces conjugate were used to target HT-29 human colorectal adenocarcinoma cells [30]. Previously, ConA-diphtheria toxin conjugate was shown to be effective against CHO and SV3T3 cultured cells and animal models [31]. ConA-daunomycin conjugate have been successfully tested on tumor-bearing mice [9]. Preliminary studies showed that the ruthenium complexes used in this study have the potential to inhibit the growth of human breast cancer cell line (ZR-75-30) (unpublished observation). In light of these observations, we propose that jacalin is extremely well suited for the binding of a variety of metal-based complexes under laboratory conditions and has the potential to be useful as vehicle for targeting such complexes to specific tissues in chemotherapeutics.
Fig. 6. Haemagglutination assays with human erythrocytes. (A) PBS buffer, (B) Jacalin, (C) galactose, (D) jacalin–galactose complex, (E) [Ru(phen)3]Cl2, (F) Jacalin– [Ru(phen)3]Cl2 complex and (G) Jacalin–galactose–[Ru(phen)3]Cl2 complex. About 100 lL of 4% human erythrocytes are used in this experiment. Jacalain agglutinates erythrocytes whereas the presence of galactose inhibits the agglutination. As inferred from this data, agglutination was only inhibited by the specific sugar galactose (D) and not by [Ru(phen)3]Cl2 (G), suggesting that [Ru(phen)3]Cl2 and galactose bind at different site. Experiments were performed in triplicate.
Conclusion In summary, this paper reports the first demonstration of the interaction of ruthenium complex with a lectin. Ruthenium complexes investigated here bind to jacalin with significantly high affinities that are comparable to the interaction of monosaccharides to this tumor-specific lectin and the interaction is mediated predominantly by hydrophobic forces. In view of the current interest in ruthenium complexes as anti-cancer agents, we envisage that a judicious use of ruthenium complex–jacalin mixtures or conjugates will be useful in increasing the drug efficacy in chemotherapeutics. Moreover, jack fruit seeds forms part of the diet in the tropics, therefore, it is also possible to administrate ruthenium–lectin complexes through oral route. Currently, studies employing cell cultures and animal models are in progress and the results will be reported in due course. Acknowledgment KBAA earnestly acknowledges the teaching assistantship from SASTRA University. VA acknowledges the financial support through Department of Science and Technology, Government of India (SB/FT/LS-217/2012). The authors thank Central Research Facility, SASTRA University for providing the necessary infrastructure. References [1] I. Ali, W.A. Wani, K. Saleem, A. Haque, Anticancer Agents Med. Chem. 13 (2013) 296–306. [2] N. Muhammad, Z. Guo, Curr. Opin. Chem. Biol. 19C (2014) 144–153. [3] (a) B.M. Blunden, A. Rawal, H. Lu, M.H. Stenzel, Macromolecules 47 (2014) 1646–1655; (b) Rademaker-Lakhai, D. van den Bongard, D. Pluim, J.H. Beijnen, J.H.M. Schellens, Clin. Cancer Res. 10 (2004) 3717–3727; (c) C. Hartinger, S. Zorbas-Seifried, M. Jakupec, B. Kynast, H. Zorbas, B. Keppler, J. Inorg. Biochem. 100 (2006) 891–904; (d) E. Alessio, G. Mestroni, A. Bergamo, G. Sava, Curr. Top. Med. Chem. 4 (2004) 1525–1535.
Table 1 Stern–Volmer quenching constant (Ksv), binding constant (Ka) and number of binding site of (n) of jacalin interacting with different ruthenium complexes. Molecule
Without 0.1 M galactose 4
Ksv 10 [Ru(phen)3]Cl2 [Ru(phen)2ppym]Cl2 [Ru(phen)2dppz]Cl2 [Ru(phen)2acdppz]Cl2
8.24 8.69 19.77 15.89
(M
1
)
With 0.1 M galactose n
Ka 10
0.98 1.06 1.00 1.08
1.86 2.27 7.08 5.62
5
1
(M
)
Ksv 104 (M1)
n
Ka 105 (M1)
9.26 6.59 13.64 12.79
1.09 0.96 0.98 1.12
2.18 3.48 6.59 4.32
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