Human serum albumin binding and cytotoxicity studies of surfactant–cobalt(III) complex containing 1,10-phenanthroline ligand

Colloids and Surfaces B: Biointerfaces 86 (2011) 35–44

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Human serum albumin binding and cytotoxicity studies of surfactant–cobalt(III) complex containing 1,10-phenanthroline ligand R. Senthil Kumar a,∗ , Preethy Paul b , A. Riyasdeen b , Georges Wagniéres a , Hubert van den Bergh a , M.A. Akbarsha c , S. Arunachalam d a

Medical Photonics Group, ISIC, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland Department of Animal Science, Bharathidasan University, Tiruchirappalli 620 024, India c Mahatma Gandhi Doerenkamp Centre for Alternatives to Use of Animals in Life Science Education, Bharathidasan University, Tiruchirappalli, India d Department of Chemistry, Bharathidasan University, Tiruchirappalli 620 024, India b

a r t i c l e

i n f o

Article history: Received 24 September 2010 Received in revised form 3 February 2011 Accepted 15 March 2011 Available online 23 March 2011 Keywords: Human serum albumin Surfactant–metal complex Cytotoxic activity Fluorescence spectroscopy Absorption optical spectroscopy

a b s t r a c t The characteristics of the binding reaction of surfactant–cobalt(III) complex, cis[Co(phen)2 (C14 H29 NH2 )]Cl2 ·3H2 O (phen = 1,10-phenanthroline, C14 H29 NH2 = tetradecylamine) with human serum albumin (HSA) were studied by fluorescence and UV–vis absorption spectroscopy. In addition, the effect of the surfactant–cobalt(III) complex on the conformation of HSA was analysed using synchronous fluorescence spectroscopy. The experimental results showed that surfactant–cobalt(III) complex caused the fluorescence quenching of HSA through a combination of static and dynamic quenching. The number of binding sites (n) and apparent binding constant (Ka ) of surfactant–cobalt(III) complex (above and below the critical micelle concentration (cmc) were determined at various temperatures. According to the thermodynamic parameters, it is likely that hydrophobic interactions are involved in the binding process. The cancer chemotherapeutic potential of surfactant–cobalt(III) complex on ME-180 cervical cancer cell was determined using MTT assay and specific staining techniques. The complex affected the viability of the cells significantly and the cells succumbed through an apoptosis process as seen in the nuclear morphology and cytoplasmic features. In addition, single-cell electrophoresis indicated DNA damage. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Human serum albumin (HSA) is an important carrier protein for several endogenous ligands such as fatty acids, bilirubin and thyroxine. It also combines with a wide variety of several small molecules including drugs, metal ions, surfactants and dye probe. Investigating the interaction of drugs with human serum albumin can elucidate the properties of drug protein complex, as it may provide useful information on the structural features that determine the therapeutic effectiveness of drugs. Interaction with albumin is also of importance to understand the drug toxicity and its distribution in the organism. It has been an interesting research field in life science, chemistry and clinical medicine [1–3]. Promising developments are emerging in regard to cobalt-based pharmaceuticals. The potential application of cobalt complexes in medicine, such as chaperones of bioactive ligands to target tumors through bio-reductive activation, has been examined over the past few years [4]. Recent drug design research in this connection has

∗ Corresponding author. Tel.: +41 0762792482. E-mail address: raj [email protected] (R.S. Kumar). 0927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2011.03.012

focused on the use of the +2 and +3 oxidation states of cobalt [5–7]. The active drug substance, bound to a chaperone cobalt complex, can be delivered to a tumor site where it is then released due the unique physiological features of the tumor. This strategy enables to avoid the unpleasant side effects of toxic anti-cancer drugs. Surfactants which are amphiphilic substances composed of both hydrophilic and hydrophobic groups are widely used in inducing unfolding of proteins and, in some special cases, stabilizing proteins at very low concentrations [8]. One important property of surfactants is the formation of micelles, which has significance in pharmaceutical science in view of their ability to increase the solubility of sparingly soluble substances in water [9] and, thus, increase their bioavailability so that they can stay in the body long enough to provide gradual accumulation in the targeted site. In addition, their sizes permit their accumulation in areas with leaky vasculature [10]. Surfactant–metal complex (i.e., metallosurfactant) is a special type of surfactant, where a coordination complex (containing a central metal ion surrounded by ligands coordinated to the metal) acts as the surfactant (Scheme 1). In these surfactants, the entity containing the central metal ion, along with its primary coordination sphere, acts as the head group and hydrophobic entity of one or

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R.S. Kumar et al. / Colloids and Surfaces B: Biointerfaces 86 (2011) 35–44

get an effective bandwidth of 5 nm. The synchronous fluorescence spectra were recorded at  = 15 and 60 nm.

hydrophilic head group

2.4. Cell culture O

S

O

O

N

N NH

hydrophobic tail group

Co

hydrophobic tail Cl

N N

Metallosurfactant

Surfactant Scheme 1.

more of the ligands acts as the tail part. Like any other well-known surfactant, for example sodium dodecyl sulfate (SDS), these metallosurfactants also form micelles above a specific concentration called a critical micelle concentration (cmc) in aqueous solution. Metal complexes of phenanthroline chelators are of great interest since they exhibit numerous biological properties such as antitumor, anti-candida and antimicrobial activities [11–13]. We have previously described our interesting results on the interaction of a few surfactant–cobalt(III) complexes containing 1,10-phenanthroline ligand with DNA and cytotoxic properties on human breast cancer cell lines [14,15]. In the present study, we report the human serum albumin interaction and cytotoxic activities of a surfactant–cobalt(III) complex containing 1,10phenanthroline against human cervical cancer cell lines. 2. Materials and methods 2.1. Materials The procedure for synthesizing the surfactant–cobalt(III) complex used in the present study is as reported in our earlier work [14]. The human breast cancer cell ME-180 was obtained from National Centre for Cell Science (NCCS), Pune, India. Human serum albumin was purchased from Sigma–Aldrich Chemie GmbH. The samples were dissolved in Tris–HCl buffer solution (0.05 M Tris, 0.15 M NaCl, pH 7.4 ± 0.1. Milli-Q water was used to prepare the solution.

ME-180 cervical cancer cells were cultured as a monolayer in Roswell Park Memorial Institute (RPMI-1640) or Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 100 ␮g/mL of streptomycin as antibiotics (Himedia, Mumbai, India), in 96 well culture plates, at 37 ◦ C, in a humidified atmosphere of 5% CO2 , in a CO2 incubator (Heraeus, Hanau, Germany). 2.5. MTT assay The surfactant–cobalt(III) complex, cis[Co(phen)2 (C14 H29 NH2 )]Cl2 ·3H2 O was dissolved in DMSO, diluted in culture medium and used to treat the chosen cell over a complex–concentration range of 5–50 ␮M for a period of 24 h and 48 h. DMSO was used as the solvent control. A miniaturized viability assay using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl2H-tetrazolium bromide (MTT) was carried out according to the method described by Mosmann [16]. The gross morphological changes in the treated cells were observed in a phase contrast microscope (Carl Zeiss, Jena, Germany) and photographed. The cells were then assayed by the addition of 20 ␮L of 5 mg/mL MTT in phosphate-buffered saline (PBS). The plates were wrapped with aluminum foil and incubated for 4 h at 37 ◦ C. The purple formazan product was dissolved by addition of 100 ␮L of 100% DMSO to each well. The absorbance was monitored at 570 nm (measurement) and 630 nm (reference) using a 96 well plate reader (Bio-Rad, Hercules, CA, USA). Data were collected for four replicates each and used to calculate the respective means. The percentage inhibition was calculated, from this data, using the formula: Mean absorbance of untreated cells (control)−Mean absorbance of treated cells×100 Mean absorbance of untreated cells (control)

The IC50 value was determined as the concentration of the complex that is required to reduce the absorbance to half that of the control. 2.6. Hoechst 33258 staining [18]

Fluorescence measurements were performed on a Perkin Elmer LS 50B Luminescence spectrometer equipped with 1.0 cm quartz cell and a thermostat bath (Varian Cary Peltier System). The absorption spectrums were recorded at room temperature on a Cary 500 UV–vis spectrophotometer equipped with 1.0 cm quartz cell.

The cell pathology was detected by staining the trypsinized cells (4.0 × 104 /mL) with 1 ␮L of Hoechst 33258 (1 mg/mL, aqueous) for 10 min at 37 ◦ C. A drop of cell suspension was placed on a glass slide and a cover-slip was laid over to reduce light diffraction. At random 300 cells were observed in a fluorescent microscope (Carl Zeiss, Jena, Germany) fitted with a 377–355 nm filter, at 400× magnification, and the percentage of cells reflecting pathological changes was calculated. Data were collected for four replicates and used to calculate the mean and the standard deviation.

2.3. Spectroscopic measurements

2.7. Acridine orange (AO) and ethidium bromide (EB) staining

Absorption titration experiments were carried out by keeping the concentration of HSA constant 10 ␮M while varying the surfactant–cobalt(III) complex concentration from 0 to 25 ␮M (below CMC) and 0 to 0.3 mM (above CMC). Fluorescence measurement was carried out at different temperatures (292, 298, 304, 310 K). The concentration of HSA was fixed at 10 ␮M and surfactant–cobalt(III) complex concentration was varied from 0 to 25 ␮M (below CMC) and 0 to 0.3 mM (above CMC). The excitation wavelength was 280 nm and the emission was monitored at 340 nm. The width of excitation and emission slits were both set to

Acridine orange and ethidium bromide staining was performed as described by Spector et al. [18]. 25 ␮L of cell suspension of each sample (both attached, released to floating by trypsinization), containing 5 × 105 cells, was treated with AO and EB solution (1 part of 100 ␮g/mL AO and 1 part of 100 ␮g/mL EB in PBS) and examined in the fluorescent microscope using an UV filter (450–490 nm). Three hundred cells per sample were counted in tetraplicate for each dose and time point. Cells were scored as viable, apoptotic or necrotic as judged by the staining, nuclear morphology and membrane integrity, and percentages of apoptotic and necrotic cells

2.2. Equipments

R.S. Kumar et al. / Colloids and Surfaces B: Biointerfaces 86 (2011) 35–44 120

37

120 Below CMC

Above CMC 100

Intensity (a.u.)

Intensity (a.u.)

100 80 60 40

80 60 40

20

20

300

350

400

450

Wavelength (nm)

300

320

340

360

380

400

420

Wavelength (nm)

Fig. 1. Emission spectra of HSA in the absence (- - -) and in the presence of surfactant–cobalt(III) complex (–). [HSA] = 10 ␮M, [surfactant–cobalt(III)] = 0–25 ␮M (below CMC) and 0–0.3 mM (above CMC). Arrow shows intensity changes upon increasing surfactant–cobalt(III) concentrations.

were then calculated. Morphological changes in the cells were also observed and photographed. 2.8. Single-cell gel electrophoresis (Comet assay) DNA damage was detected by adopting the Comet assay of Singh et al. [19]. The cells were treated with the surfactant–cobalt(III) complex for 24 and 48 h. The harvested cells were suspended in low-melting-point agarose in PBS and pipetted out to microscope slides pre-coated with a layer of normal-melting-point agarose. Slides were chilled on ice for 10 min and then immersed in lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, 0.2 mM NaOH, pH 10.01 and Triton X-100) and the solution was kept

Fig. 2. Stern–Volmer and modified Stern–Volmer plots of surfactant–cobalt(III) complex below and above CMC (T = 292 K) with HSA.

overnight at 4 ◦ C in order to lyse the cells and to permit DNA unfolding. Thereafter, the slides were exposed to alkaline buffer (300 mM NaOH, 1 mM Na2 EDTA, pH > 13) for 20 min to allow DNA unwinding. The slides were washed with buffer (0.4 M Tris, pH 7.5) to neutralize excess alkali and to remove detergents before staining with ethidium bromide (20 ␮L in 50 ␮g/mL). Photomicrographs were obtained using the fluorescent microscope. 150 cells from each treatment group were digitalized and analysed using CASP software. The images were used to estimate the DNA content of individual nuclei and to evaluate the degree of DNA damage representing the fraction of total DNA in the tail.

Fig. 3. Van’t Hoff plot of HSA–surfactant–cobalt(III) complex below and above CMC.

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R.S. Kumar et al. / Colloids and Surfaces B: Biointerfaces 86 (2011) 35–44 15 Below CMC

0,4

Intensity (a.u.)

3

320

360

Above CMC B

2,4 2,0

A

120

1,6 1,2

80

0,8 40

Absorbance

0,8

6

Absorbance

1,2 B

Intensity (a.u.)

A

12 9

160

1,6

0,4

400

320

Wavelength (nm)

360

400

440

Wavelength (nm)

Fig. 4. Spectral overlap between the fluorescence emission spectrum of HSA (A) and UV–vis absorption spectrum of surfactant–cobalt(III) complex (B). [surfactant–cobalt(III)] = [HSA] = 1 ␮M (below CMC) and 1 mM (above CMC).

3. Results and discussion 3.1. Fluorescence studies of HSA quenched by surfactant–cobalt(III) complex Fluorescence quenching refers to any process that decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in fluorescence quenching of excited state fluorophores. These include molecular rearrangement, energy transfer, ground state complex formation and collisional quenching. The effect of surfactant–cobalt(III) complex on HSA fluorescence intensity is shown in Fig. 1. As shown in this figure, the addition of surfactant–cobalt(III) complex led to a concentrationdependent quenching of HSA intrinsic fluorescence intensity, implying that the binding of surfactant–cobalt(III) complex to HSA. Quenching can occur by different mechanisms, which usually classified as dynamic quenching and static quenching. Dynamic and static quenching can be distinguished by their different temperature dependence of binding constants and viscosity. Generally, the quenching constants decrease with increasing temperature for static quenching, but the reverse effect is for dynamic quenching [20,21]. For dynamic quenching, the mechanism can be described by the Stern–Volmer equation [20]: F0 = 1 + Ksv [Q ] F

(1)

where F0 and F are the fluorescence intensities of HSA in the absence and presence of the quencher, respectively. Ksv is the Stern–Volmer quenching constant, and [Q] is the concentration of quencher.

Stern–Volmer plots of HSA with surfactant–cobalt(III) complex below CMC and above CMC are shown in Fig. 2. Below CMC, the Stern–Volmer plot follows a linear relation whereas, above CMC, the plot shows a positive deviation (concave towards the y axis), suggesting the presence of a combination of static and dynamic quenching. According to Eftink and Ghiron [22,23], the upward curvature in the Stern–Volmer plot indicates that an additional static quenching takes place near the subdomain(s) where tryptophan contained in the HSA residues are located. Thus, the positive deviation above CMC is due to the presence of a quencher molecule in the micellar cage at the moment of fluorescence excitation. In order to calculate the Ksv value of the surfactant–cobalt(III) complex above CMC, a modified Stern–Volmer equation that describes quenching data for both dynamic and static quenching was applied by Lakowicz [20]: F0 = 1 + Ksv [Q ] exp(V [Q ]) F

(2)

where V is the static quenching constant. This value of V can be obtained from Eq. (2) by plotting F0 /F exp(V[Q]) − 1 versus [Q], for differing V, until a linear plot is acquired. The Ksv can then be obtained from slope of F0 /F exp(V[Q]) − 1 versus [Q] plot. Values of V and Ksv at four different temperatures (292, 298, 304 and 310 K) are consequently presented in Table 1. The Stern–Volmer quenching constant increases with increasing temperature below CMC, while it decreases above CMC. As for a bimolecular quenching process, Eq. (3) is employed for the evaluation of the quenching rate constant kq : kq =

Ksv 0

(3)

Fig. 5. Absorption spectra of HSA in the absence (- - -) and in the presence of increasing amounts of surfactant–cobalt(III) complex (–). [HSA] = 10 ␮M, [surfactant–cobalt(III)] = 2–25 ␮M (below CMC) and 0–0.3 mM (above CMC). Arrow shows the absorbance changes upon increasing surfactant–cobalt(III) complex concentrations.

R.S. Kumar et al. / Colloids and Surfaces B: Biointerfaces 86 (2011) 35–44

39

Table 1 Stern–Volmer quenching constants for the interaction of surfactant–cobalt(III) complex with HSA at different temperatures. T (K)

Ksv (L mol−1 )

kq (L mol−1 s−1 ) 4

292 298 304 310

3

V (L mol−1 ) 112

Below CMC (×10 )

Above CMC (×10 )

Below CMC (×10

6.54 7.46 7.86 8.23

3.74 2.87 2.29 1.65

6.54 7.46 7.86 8.23

11

)

Ra 3

Above CMC (×10 )

Above CMC (×10 )

Below CMC

Above CMC

3.74 2.87 2.29 1.65

9.07 8.88 8.65 8.59

0.9985 0.9937 0.9921 0.9922

0.9949 0.9953 0.9998 0.9901

Ra – correlation coefficient. Table 2 Binding constants and number of binding sites of surfactant–cobalt(III) complex–HSA at different temperatures. T (K)

Ka (L mol−1 )

292 298 304 310

Ra

n 5

7

Below CMC (×10 )

Above CMC (×10 )

Below CMC

Above CMC

Below CMC

Above CMC

0.69 0.91 1.19 1.63

2.83 3.89 5.62 8.37

1.006 1.019 1.039 1.064

1.822 1.863 1.911 1.959

0.9989 0.9954 0.9958 0.9959

0.9993 0.9936 0.9951 0.9916

Ra – correlation coefficient for the van’t Hoff plot.

Table 3 Thermodynamic parameters for the binding of surfactant–cobalt(III) complex to HSA. T (K)

292 298 304 310

H (KJ mol−1 )

G (KJ mol−1 )

Below CMC

Above CMC

35.76

45.36

Below CMC

Above CMC

−27.06 −28.28 −29.55 −30.94

−41.66 −43.29 −45.10 −47.02

where kq is the quenching rate constant of the biomolecule, and  0 is the average lifetime of the biomolecule without quencher. The results showed that the rate constants for the quenching of HSA caused by surfactant–cobalt(III) complex were higher than the limiting collisional quenching rate constant of the biomolecule (2 × 1010 L mol−1 s−1 ) [24,25], which suggests that the overall quenching is dominated by static quenching. 3.2. Analysis of binding equilibria The binding constant (Ka ) and the number of binding sites (n) can be determined according to the method described by Chipman et al. [26], using the equation log

S (J mol−1 K−1 )

F0 − F = log Ka + n log[Q ] F

Below CMC

Above CMC

215

297.79

tion of the surfactant–cobalt(III) complex above CMC. Recently, we have reported that the CMC values of the surfactant–cobalt(III) complexes increase with increasing the temperature [14,15]. This behavior may be related to two competitive effects. Firstly, a temperature increase causes a decrease in hydration in the hydrophilic group, which favors micellization. Secondly, a temperature increase also disrupts the water surrounding the hydrophobic group, and this retards micellization. The relative magnitude of these two opposing effect determine the CMC behavior. The number of binding sites (n) below CMC is approximately equal to 1, indicating that there is only one class of binding site, whereas, the value of n is approximately 2, above CMC which indicates that there is more than one class of binding site to surfactant–cobalt(III) complex in HSA.

(4)

where Ka is apparent binding content of surfactant–cobalt(III) complex with HSA and n is the number of binding sites per albumin molecule, which can be determined by the slope and the intercept of double logarithm regression curve of log ((F0 –F)/F) versus log [Q] based on Eq. (4). We have calculated the apparent binding constant and number of binding site values for our surfactant–cobalt(III) complex below CMC and above CMC. The values are shown in Table 2. The table shows that the binding constants below CMC are lower than those of the corresponding constants above CMC and also the binding constant value increases with increasing temperature for a given system. This is because of the higher hydrophobicity, and the micelle forma-

3.3. Determination of the force acting between surfactant–cobalt(III) complex and HSA

Table 4 Inhibition of in vitro cancer cells growth by surfactant–cobalt(III) complex.

ln K = −

Surfactant–cobalt(III) complexes

cis-[Co(phen)2 (C14 H29 NH2 )Cl](ClO4 )2

IC50 (␮M) 24 h

48 h

28 ± 3

16 ± 2

Thermodynamic parameters relying on temperatures were analysed to characterize the acting force between drug molecules and biomacromolecules. There are essentially four types of noncovalent interactions that could play a key role in drug binding interaction to proteins. These are hydrophobic forces, van der Waals forces, electrostatic interactions and hydrogen bonds [27]. In order to elucidate the interaction of surfactant–cobalt(III) complex with HSA, the thermodynamic parameters were calculated from the van’t Hoff equation: H o S o + RT R

(5)

where K is the analog to the associative binding constants corresponding to various temperatures, and R is the gas constant. The enthalpy change (Ho ) can be calculated from the slope of the van’t Hoff relationship (Fig. 3). The free energy change (Go ) was

40

R.S. Kumar et al. / Colloids and Surfaces B: Biointerfaces 86 (2011) 35–44

35

35 Below CMC

Above CMC

30

30

25

25

Intensity (a.u.)

Intensity (a.u.)

a

20 15

20 15

10

10

5

5

280

300

280

Wavelength (nm)

b

120

120

Above CMC

Below CMC

100

Intensity (a.u.)

100

Intensity (a.u.)

300

Wavelength (nm)

80 60

80 60

40

40

20

20

260

280

260

300

280

300

Wavelength (nm)

Wavelength (nm)

Fig. 6. Synchronous fluorescence spectrum of HSA with  = 15 nm (a) and 60 nm (b) in the absence (- - -) and in the presence of increasing amounts of surfactant–cobalt(III) complex (–). [HSA] = 10 ␮M, [surfactant–cobalt(III)] = 0–25 ␮M (below CMC) and 0–0.3 mM (above CMC). Arrow shows the intensity changes upon increasing surfactant–cobalt(III) complex concentrations.

plex) and R0 is the critical distance when their transfer efficiency is 50%. It is given by the following equation:

consequently obtained according to the following equation: Go = H o − TS o = −RT ln K

(6)

The thermodynamic parameters for the interaction of surfactant–cobalt(III) complex with HSA are shown in Table 3. The negative sign for G means that the interaction process is spontaneous. The positive H and S values indicate that hydrophobic force may play a major role in the binding between surfactant–cobalt(III) complex and HSA [27].

R06 = 8.8 × 10−25 k2 N −4 ˚J

where k2 is the special orientation factor of the dipole, N the refractive index of the medium, ˚ the fluorescence quantum yield of the donor and J is the overlap integral of the fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. J is given by, J=

3.4. Energy transfer from HSA to surfactant–cobalt(III) complex The importance of the Förster resonance energy transfer in biochemistry is that the efficiency of energy transfer can be used to evaluate the distance between the drug and the tryptophan residues responsible for the natural intrinsic fluorescence of the protein. The binding distance (r) between a protein residue (donor) and a bound drug molecule (acceptor) can be calculated from the Förster’s non-radiative energy transfer (FRET) theory [28]. The efficiency of energy transfer (E) is related to the distance (R0 ) between donor and acceptor by: E=−

R6 + r 6 F = 0 6 F0 R0

(7)

(8)

˙F()ε()4  ˙F()

(9)

where F() is the fluorescence intensity of the fluorescence donor of wavelength, , and ε () is the molar absorption coefficient of the acceptor at wavelength, . The overlap of the UV absorption spectrum of surfactant–cobalt(III) complex with the fluorescence emission spectrum of the HSA is shown in Fig. 4. In the present case, k2 = 2/3, N = 1.336 and ˚ = 0.118 for HSA [29]. From Eq. (7)–(9), we could calculate that J = 4.32 × 10−14 M−1 cm3 , E = 0.620, R0 = 3.13 nm and r = 3.00 nm (below CMC) and J = 1.22 × 10−16 M−1 cm3 , E = 0.897, R0 = 1.18 nm and r = 0.85 nm (above CMC). The distance between donor and acceptor is less than 8 nm [30], which implies that the energy transfer from HSA to surfactant–cobalt(III) complex occurred with high probability, especially above CMC. 3.5. Absorption spectroscopic studies

where F and F0 are the fluorescence intensities of HSA in the presence and absence of surfactant–cobalt(III) complex, r is the distance between donor (HSA) and acceptor (surfactant–cobalt(III) com-

UV–vis absorption spectroscopic measurement is a simple and effective method to explore the structural change and to estab-

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41

Fig. 7. Hoechst 33258-stained ME-180 cervical cancer cells treated with surfactant–cobalt(III) complex for 24 and 48 h. 400×; C and (a) control; (b) necrotic cells; (c) and (d) early apoptotic cells; (e) and (f) late apoptotic cells; (g) and (h) apoptotic cell death final event apoptotic bodies formation.

lish the complex formation [31]. The absorption spectra of HSA in the absence and in the presence of surfactant–cobalt(III) complex (below and above CMC) are shown in Fig. 5. With increasing concentration of surfactant–cobalt(III) complex, the absorption bands of HSA were affected, resulting in the tendency of hyperchromism and a slight blue shift. The results indicated that there exists interaction between surfactant–cobalt(III) complex and HSA through ground state complex formation. The blue shift expresses that the conformation of HSA has been changed. 3.6. Conformational investigation Synchronous fluorescence spectra can be used to analyse conformational changes in HSA. It gives information about the molecular environment in the vicinity of the chromophores molecules and has several advantages, such as a high sensitivity, spectral simplification and avoiding different perturbing effects [24]. Synchronous fluorescence spectra were obtained by simultaneously scanning excitation and emission monochromators. As  between excitation wavelength and emission wavelength is 15 nm, synchronous fluorescence offers characteristics of tyrosine residues, while when  is 60 nm, it provides the characteristic information of tryptophan residues. Synchronous fluorescence spectra of HSA upon addition of surfactant–cobalt(III) complex (below and above CMC) at  = 15 and 60 nm are shown in Fig. 6. As shown in this figure, the quenching of the fluorescence intensity of tryptophan residues is stronger that that of the tyrosine residues, suggesting that tryptophan residues contribute greatly to the quenching of the intrinsic

fluorescence. Moreover, a red shift of the maximum emission wavelength of tyrosine and tryptophan residues from 286 to 288 nm at below CMC) and 286–290 nm at above CMC, indicates that the conformation of HSA changed and the hydrophobicity around both the residues increased. 3.7. Effect on the viability of cells The cytotoxic activity of surfactant–cobalt(III) complex was examined on cultured ME-180 human cervical cancer cell lines by exposing cells for 24 and 48 h to the medium containing the complex at 5–50 ␮M concentrations and using MTT assay. The surfactant–cobalt(III) complex inhibited the growth of cervical cancer cells significantly in a dose- and duration-dependent manner. The cytotoxic activity was determined according to the dose values of the exposure of the complex required to reduce survival of the cell to 50% (IC50 ). The IC50 values obtained in this study are given in Table 4. The surfactant–cobalt(III) complex showed high cytotoxic activity against the cervical cancer cell. The cytotoxic effect of the complex could be due to their amphiphilic nature by which they have the capacity to penetrate the cell membrane easily, reduce the energy status in tumors and also to alter hypoxia status in the cancer cell microenvironment, which are factors which would determine the anti-tumor activity [32]. There is evidence that cobalt complexes cause activation of lipid peroxidation, DNA damage, and reduction of the bioenergetic status of tumor tissues [5]. It may be also concluded that the level of cellular damage inflicted by this complex depends on the nature of their axial lig-

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Fig. 8. Photomicrographs of control (C) and AO and EB stained ME-180 cancer cells treated with surfactant–cobalt(III) complex for 24 and 48 h; 400× (a) control; (b) necrotic cells; (c)–(h) morphological changes in apoptotic cells.

ands. It is known that phenanthroline-containing metal complexes have a wide range of biological activities such as antifungal, apoptotic [33,34], interaction with DNA thereby inhibiting replication, transcription and other nuclear functions and arresting cancer cell proliferation so as to arrest tumor growth [35]. 3.8. Cell death as revealed in fluorescent staining Apoptosis is characterized by DNA fragmentation, chromatin condensation and marginalization, membrane blebbing, cell shrinkage, and fragmentation of the cells into membrane-enclosed vesicles or apoptotic bodies, to be phagocytosed by macrophages. To detect apoptosis at a basic level, we adopted Hoechst staining, which reveals the changes in the gross cytology of the cell, with special reference to cytoplasm and nucleus [18]. After treatment of ME-180 cervical cancer cells with surfactant–cobalt(III) complex at the IC50 concentration for 24 and 48 h, the cells were observed for the gross cytological changes. The treated cells revealed the microscopic cytological changes characteristic of apoptosis listed above and also late apoptosis indication of dot-like chromatin. However, a few cells indicated features of necrotic death (Fig. 7). Additional microscopic evidence for apoptosis was obtained using acridine orange (AO) and ethidium bromide (EB) staining, the fluorescence patterns of which depend upon viability and membrane integrity of the cells [36]. Uniformly green fluorescing nuclei with a highly organized structure indicated normal and viable cells (Fig. 8). Green fluorescing nuclei, with peri-nuclear chromatin condensation as revealed in bright green patches or fragments, indicated early apoptotic cells (Fig. 8). Orange to red fluorescing

nuclei, with highly condensed or fragmented chromatin indicated late apoptotic cells (Fig. 8). Uniformly orange to red fluorescing nuclei with no indication of chromatin fragmentation but the entire cells as well as nuclei were swollen to large size indicated necrotic cells (Fig. 8). Data on cells indicating apoptotic and necrotic morphologies, induced by the treatments with various concentrations of surfactant–cobalt(III) complex for 24 and 48 h, followed by staining with AO and EB, collected from manual counting of cells, are presented in Fig. 9, which reveal that the complex is efficient in inducing apoptosis to a great extent but also necrosis to a certain extent, in the cervical cancer cells.

Fig. 9. 100% stacked column of percent normal, necrotic and apoptotic cells as revealed by AO and EB staining followed by manual counting. Cells treated with surfactant–cobalt(III) complex.

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Fig. 10. Surfactant–cobalt(III) complex treated ME-180 cervical cancer cells as revealed in the comet assay. Comet images of DNA double strand breaks at 12 and 24 h treatment of surfactant–cobalt(III) complex.

3.9. DNA damage as revealed in single cell gel electrophoresis (Comet assay) Among the different techniques adopted for measuring and analysing DNA-strand breaks typical of toxic DNA damage and of early stage of apoptosis in mammalian cells [36], single cell gel electrophoresis assay (comet assay) is considered to be rapid, simple, visual and sensitive. In the comet assay the DNA of the damaged cell takes the appearance of a comet, with head and tail regions.

The CASP image analysis software is of help in (i) the analysis of a variety of geometric and densitometry parameters, and (ii) estimation of the amount of DNA in the head (intact DNA) and the tail (DNA with strand breaks) regions (Fig. 10). Since the tail length and density reflect the extent of strand breaks in the DNA, the percentage of DNA in the tail provides a quantitative measure of the damaged DNA as fraction of the total DNA. The results reveal that DNA damage was induced in cervical cancer cells by the surfactant–cobalt(III) complex at 24 and 48 h, in a duration-dependent manner but, as shown in Fig. 11, the complex produced a relatively higher percentage of damaged cells at 12 h than at 24 h. DNA damage is an early event in programmed cell death and, thus, the DNA in the comet tail is suggesting an entry of cells into apoptosis [37]. Our results from the comet assay match morphological evidences of apoptosis. Here, the different degrees of DNA damage may be due to structural differences in the ligands.

4. Conclusion

Fig. 11. DNA damage in ME-180 cervical cells populations as defined according to the percentage of DNA in the tail: the multiple parts of each columns represent (from the bottom to the top) intact (0–20%), slightly damaged (20–40%), damaged (40–60%), highly damaged (60–80%) and dead (80–100%).

The interaction between HSA and surfactant–cobalt(III) complex, cis-[Co(phen)2 (C14 H29 NH2 )]Cl2 ·3H2 O has been investigated using fluorescence and absorption spectroscopic techniques. The results obtained give preliminary information on the binding of surfactant–cobalt(III) complex to HSA. HSA molecules have a relatively high affinity with surfactant–cobalt(III) complex, through hydrophobic interaction. The results of synchronous

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fluorescence spectroscopy and UV–vis absorption spectroscopy indicate that the microenvironment of HSA are disturbed by the surfactant–cobalt(III) complex. The surfactant–cobalt(III) complex produced evidence of affecting viability and inducing cell death, predominantly apoptosis, in ME-180 cervical cancer cells. The necrotic death of cells, occurring on certain circumstances, is also a desired end point in cancer therapy. From these perspectives, it would be pertinent to extend the study further to find the mechanism(s) underlying the cell death by finding the expression of proand anti-apoptotic genes and caspases in the case of apoptosis, and oxidative damage in the case of necrosis. Acknowledgement The authors gratefully acknowledge a fellowship for Senthil Kumar from the Federal Commission for Scholarships for Foreign Students (FCS) in Switzerland. References [1] Y.Q. Wang, H.M. Zhang, H.Q. Zhou, J. Mol. Struct. 932 (2009) 31. [2] F. Ge, C. Chen, D. Liu, B. Han, X. Xiong, S. Zhao, J. Lumin. 130 (2010) 168. [3] S.M. Darwish, S.E. Abu sharkh, M.M. Abu Teir, S.A. Makharza, J. Mol. Struct. 963 (2010) 122. [4] I.H. Hall, C.B. Lackey, T.D. Kistler, R.W. Durham, J.M. Russell, R.N. Grimes, Anticancer Res. 20 (2000) 2345. [5] S. Osinsky, I. Levitin, L. Bubnovskaya, A. Sigan, I. Ganusevich, A. Kovelskaya, N. Valkovskaya, L. Campanella, P. Wardman, Exp. Oncol. 26 (2004) 140. [6] R. Senthil Kumar, S. Arunachalam, V.S. Periasamy, C.P. Preethy, A. Riyasdeen, M.A. Akbarsha, Polyhedron 28 (2008) 1111. [7] B.A. Teicher, M.J. Abrams, K.W. Rosbe, T.S. Herman, Cancer Res. 50 (1990) 6971. [8] K.P. Ananthapadmanabhan, in: E.D. Goddard, K.P. Ananthapadmanabhan (Eds.), Interactions of Surfactants with Polymers and Proteins, first ed., CRC Press, London, 1993. [9] S. Mall, G. Buckton, D.A. Rawlins, J. Pharm. Sci. 85 (1996) 75. [10] V.P. Torchilin, J. Control. Release 73 (2001) 137.

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