Download Concentration Dependent Different Action of Tamoxifen on

Biosci Rep (2007) 27:247–255
DOI 10.1007/s10540-007-9050-3
ORIGINAL PAPER
Concentration Dependent Different Action
of Tamoxifen on Membrane Fluidity
Nadide Kazanci Æ Feride Severcan
Published online: 28 June 2007
The Biochemical Society 2007
Abstract Tamoxifen (TAM) is a non-steroidal antiestrogen drug, which is widely used
to prevent and treat breast, liver, pancreas and brain cancers. The present work
investigates, in detail, the concentration dependent behavior of TAM (varying from
1 mol% to 45 mol%) on membrane fluidity. The differential scanning calorimetry
(DSC) studies showed that tamoxifen eliminates the pre-transition and decreases the
main phase transition to lower temperatures. Using visible spectroscopy at 440 nm and
Fourier transform infrared (FTIR) spectroscopy it was found that membrane dynamics
decreases for 1 and 3 mol% tamoxifen in both the gel and liquid crystalline phases.
Above these concentrations up to 18–24 mol%, it increases and reaches its maximum
values. As tamoxifen concentration was further increased, the membrane dynamics is
found to be gradually decreased, although TAM still has fluidifying effect in comparison
to pure phospholipid membrane. These findings are important for the effective use of
tamoxifen in the cancer therapy to eliminate its dose dependent side effects reported in
the literature.
Keywords Tamoxifen Model membrane DPPC Liposome Membrane fluidity Lipid phase transition FTIR spectroscopy DSC Visible spectroscopy
Introduction
At therapeutic doses, drugs elicit several responses, some of which cause unwanted side
effects (De Lima et al. 2003). Although drugs follow several routes of administration
N. Kazanci
Department of Physics, Faculty of Science, Ege University, Izmir 35100, Turkey
F. Severcan (&)
Department of Biology, Middle East Technical University, Ankara 06531, Turkey
e-mail: [email protected]
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before they are given to patients, their interactions with macromolecular components of
human body are generally incomplete. Since biological membranes contain different
type of lipids, proteins, and polysaccharides, it is difficult to understand the mechanism
of drug-lipid interactions in biological systems. Therefore, studies are firstly concentrated on drug-model membrane interactions (Toyran and Severcan 2003; Severcan
et al. 2005; Gagoś et al. 2004). Tamoxifen (TAM) is a non-steroidal antiestrogen drug,
which is widely used to prevent and treat breast, liver, pancreas and brain cancers
(Wisemanand Tamoxifen 1994). It is not clear whether tamoxifen’s action is due to a
direct interaction with relevant receptors or enzymes, or due to the consequences of its
interaction with cellular membrane (Engelke et al. 2001; Custodio 1993; Wiseman et al.
1993; Boyar and Severcan 1997; Dicko et al. 1999; Severcan et al. 2000; Engelke et al.
2002; Lehenkari et al. 2003; McDermott et al. 1998; Gao and Dluzen 2001). Previous
studies on artificial and biological membranes showed that TAM locates in the
hydrophobic part of the membrane and affects the physical properties (Custodio 1993;
Severcan et al. 2000; Luxo et al. 1996) and chemical composition of lipid bilayers (Luxo
et al. 1996). TAM molecules can accumulate in biomembranes and even produce high
local concentrations (Lehenkari et al. 2003).
Hydrophobic interactions, membrane fluidity, and drug lipophilicity are decisive for
efficient intracellular uptake of anticancer drugs (Jedrzejczak et al. 1999; Schuldes et al.
2001). Additionally, membrane fluidity studies have also been considered as a promising
approach for cancer therapy (Ferreira et al. 2005). It was previously reported that
membrane fluidity is directly correlated with the metastatic capacity of some murine
tumor cell lines (Taraboletti et al. 1989).
The studies regarding the effect of tamoxifen on membrane fluidity are limited and
contradictory to each other (Engelke et al. 2001; Custodio 1993; Wiseman et al. 1993;
Dicko et al. 1999; Severcan et al. 2000). We have previously performed a Fourier
Transform Infrared Spectroscopic (FTIR) study and showed the different action of
TAM on membrane fluidity at low (1 mol%) and high (30 mol%) TAM concentrations
(Severcan et al. 2000). However we do not know the effect of tamoxifen on membrane
fluidity at other concentrations. Therefore in the current study, we aimed to better
understand the concentration dependent action of TAM on membrane dynamics. A
zwitterionic membrane made of dipalmitoyl phosphatidylcholine (DPPC) was chosen as
a model to simplify the system for better elucidating the drug-lipid interactions. This is
important for the effective using of the drug, because adverse effects of TAM therapy
were previously reported as dose-related (De Lima et al. 2003).
Materials and Methods
TAM and DPPC were purchased from Sigma (St. Louise, Mo) and used without further
purification (Severcan et al. 2000).
For turbidity studies pure phospholipid multilamellar liposomes (MLV) were
prepared according to the procedure reported in Severcan et al. (1995). About 1.5 mg
of lipid was dissolved in chloroform. Residual solvent was removed by subjecting the
films to vacuum drying in a spin-vacuum (HETO-spin vac) system for two hours. Dry
thin film was hydrated with 1.5 ml of 10 mM phosphate buffer (pH 7.4). Multilamellar
liposomes were formed by vortexing the mixture for 20 min at a temperature that is
20C above the main phase transition temperature of the lipid. TAM -containing
liposomes were prepared with exactly the same procedure, except that the desired
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amount of tamoxifen dissolved in ethanol was firstly put into the round-bottomed flask.
The details were reported in Severcan et al. (1995). Turbidity studies were carried out
using a Varian-Cary 300 UV/Visible spectrometer. Turbidity studies are considered as a
special form of light scattering experiments (Freifelder 1982).
The lipid mixture for infrared measurements was prepared by a procedure similar to
the procedure mentioned above for turbidity studies except that this time 5 mg lipid was
hydrated with 25 ll phosphate buffer as reported in Severcan et al. (2005). Infrared
spectra were obtained using a BOMEM, MB-157 FTIR spectrometer. About 20 ll of
sample suspension was placed between CaF2 windows, separated by a 12 lm Mylar
spacer. Interferograms were averaged for 100 scans at 2 cm–1 resolution. The temperature was regulated by a Unicam Specac digital temperature controller unit and a
thermocouple located on the edge of the cell window. The samples were incubated for
10 min at each temperature before spectral acquisition. The details of the spectral
analysis were reported in Korkmaz and Severcan (Korkmaz and Severcan 2005).
The lipid mixture for the DSC measurements were prepared according to the same
procedure as for the infrared study; however, thin films were obtained this time by
hydrating 2 mg of phospholipids with 50 ll phosphate buffer (Severcan et al. 2005;
Korkmaz and Severcan 2005). Experiments were performed with a Universal TA DSC
Q 100 V6.21 instrument. The samples were encapsulated in hermatically sealed standard
DSC pans. An empty pan was used as reference during the measurements to exclude the
calorimetric effect of the pan automatically by the associated computer program.
Heating rate was 0.2C/min. Calorimetric investigation was carried out in a temperature
range of 25–60C.
Results and Discussion
The effect of TAM on membrane phase behavior was determined by DSC technique
(Fig. 1). As seen from the figure the well known DPPC phase transition occurs at
40.99C and pre-transition occurs at 35C which are in agreement with previous studies
(Korkmaz and Severcan 2005; Kazancı et al. 2001). The presence of TAM in DPPC
liposomes leads to a broadening of the transition profile and lowering of the main phase
transition temperature, in agreement with previous DSC studies performed on DPPC
liposomes in the presence of 0–7 mol% TAM (Engelke et al. 2001). The main phase
transition temperature decreased around 0.4–2C depending on TAM concentration.
We also observed that TAM eliminates pre-transition of DPPC liposomes.
Fluidity is a physical parameter susceptible to perturbations by membrane active
drugs (Severcan et al. 2005; Monteiro et al. 2003). In the current study, we investigated
membrane fluidity at 29 and 55C, which monitor the gel and liquid crystalline phase of
the membrane, respectively as seen from the DSC studies. To achieve this, the
bandwidth of CH2 antisymmetric stretching vibrational modes of DPPC liposomes
(Toyran and Severcan 2003; Severcan et al. 2005) and absorbance values at 440 nm
(Severcan et al. 2000, 1995; Chong and Colbow 1976; Stillwell and Wassall 1990; Viti
et al. 1985 ; Ohki and Duzgunes 1979) were studied. All experiments were repeated five
times and similar trends were observed at each repeat.
Figure 2 shows the average spectra in the 3000–2800 cm–1 region for pure DPPC
liposomes in the absence and presence of low (1 mol%) and high (30 mol%)
concentrations of tamoxifen. The spectra were normalized with respect to the CH2
antisymmetric stretching band. The most intense bands in the infrared spectra of a
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45mol%tam+dppc
30mol%tam+dppc
Endothermic Heat Flow
24mol%tam+dppc
18mol%tam+dppc
12mol%tam+dppc
9mol%tam+dppc
6mol%tam+dppc
3mol%tam+dppc
1mol%tam+dppc
dppc
30
32
34
36
38
40
42
44
Temperature (°C)
Fig. 1 DSC thermograms of DPPC liposomes in the absence and presence of different concentration of
tamoxifen
Fig. 2 The average spectra for pure DPPC liposomes in the absence and presence of low (1 mol%) and
high (30 mol%) concentrations of tamoxifen in the 3000–2800 cm–1 region. Spectra were normalized
with respect to the CH2 antisymmetric stretching band
phospholipid model membrane are due to methylene antisymmetric and symmetric
stretching vibrations, which appear around 2920 cm–1 and 2850 cm–1, respectively. The
bandwidth of these bands is sensitive to molecular motion and therefore is used to probe
lipid fluidity in membranes (Toyran and Severcan 2003; Severcan et al. 2005; Korkmaz
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and Severcan 2005). The different variations in the bandwidth for low and high TAM
concentrations are clearly seen in the figure.
Figure 3 shows the concentration dependence of the bandwidth of the CH2
antisymmetric stretching bands of DPPC liposomes in the absence and presence of
TAM at 29 and 55C. As previously mentioned, these temperatures were chosen to
monitor the gel and liquid crystalline phases of the DPPC membrane, respectively. As
can be seen from Fig. 2, the vibrational band of CH2 antisymmetric stretching mode was
sufficiently separated after careful water subtraction procedure; therefore, it was not
necessary to use band deconvolution or fit routines to evaluate their bandwidths for
relative measurements for this model membrane study, as reported by others (Toyran
and Severcan 2003; Severcan et al. 2005; Korkmaz and Severcan 2005). Bandwidth was
measured at 0.75· peak height position. Qualitatively similar results were also obtained
at 0.50· peak height position (not shown). As seen from Fig. 3, with the addition of 1
and 3 mol% TAM the bandwidth decreases. Above 3 mol% TAM concentrations, the
bandwidth starts to increase and above 6 mol% the bandwidth increases with respect to
that of pure DPPC. It reaches its maximum value between 18 and 24 mol% TAM
concentrations indicating an increase in lipid dynamics (Toyran and Severcan 2003;
Severcan et al. 2005; Korkmaz and Severcan 2005). Above these values, the bandwidth
stars to decrease as TAM concentration increases. It almost reaches the value of pure
DPPC at a very high TAM concentration (45 mol%). Fig. 4
These results are further supported by visible spectroscopic studies at 440 nm. This
wavelength value was chosen to minimize light scattering (Chong and Colbow 1976).
The decrease in absorbance reflects a decrease in aggregation and fusion among the
vesicles indicating a decrease in particle size and therefore both phenomena cause a
decrease in absorbance (Ohki and Duzgunes 1979). However, the phase transition of
phospholipids from the gel to the liquid crystalline phase also causes a decrease in
absorbance values. This is mainly due to the changes that occur in the refractive index of
the lipids, as a consequence of changes in the lipid density during melting (Yi and
MacDonald 1973). Figure 3 shows the temperature dependent behavior of the
absorbance values at 440 nm for DPPC liposomes in the absence and presence of
different concentrations of TAM. As can be seen from the figure an opposite behavior
of the absorbance values for low and high TAM concentrations was observed. The
24
CH2 Antisymmetric Stretching
29 ºC
55 ºC
Bandwidth (cm-1)
22
20
18
16
14
12
10
0
5
10
15
20
25
30
35
40
45
50
Concentration (mol %)
Fig. 3 The temperature dependence of the bandwidth of the CH2 antisymmetric stretching bands in the
infrared spectra of DPPC liposomes in absence and presence of different tamoxifen concentrations
varying at 29C and 55C
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1
DPPC
DPPC+12 mol%TAM
DPPC+1 mol%TAM
DPPC+18 mol%TAM
DPPC+3 mol%TAM
DPPC+30 mol%TAM
DPPC+6 mol%TAM
DPPC+ 45 mol%TAM
0.9
Absorbance
0.8
0.7
0.6
0.5
0.4
0.3
0.2
25
30
35
40
45
50
55
60
65
70
Temperature (oC)
Fig. 4 Temperature dependence of absorbance at 440 nm for DPPC liposomes in the absence and
presence of different concentrations of tamoxifen
absorbance increases for low TAM concentrations (1 and 3 mol%) indicates a decrease
in lipid fluidity (Severcan et al. 2000). Whilst it decreases for higher TAM concentrations, indicating an increase in lipid fluidity. Similar to FTIR studies, at very high TAM
concentrations (e.g. 45 mol%), the absorbance value increases and approaches to the
values of pure DPPC.
TAM and derivatives have been shown to protect biological membranes, and
membrane-model systems such as liposomes, against free radical mediated lipid peroxidation. Observation of this protective action of TAM and its derivatives can be attributed
to molecular modulation of the membrane environment by the interaction of sterols with
the particular fatty acid side-chain present in membrane phospholipids (Wiseman 1994;
Moreira et al. 2004). It is suggested that most of these drugs cause their toxic effects
through incorporation into cell membranes. Therefore studies of the interactions of drugs
with biomembranes are important in understanding the mechanism of their action.
There are conflicting results on the effect of TAM on membrane dynamics. Most of
the studies reported the stabilizing effect of it on membranes, by restricting the mobility
of the lipids (Wiseman et al. 1993; Boyar and Severcan 1997; Wiseman 1993; Wiseman
et al. 1992; Wiseman et al. 1993). In contrast, an increase in membrane fluidity
modulated by TAM was recently reported (Dicko et al. 1999). However, in other study
the effects of TAM on liposomes of phospholipids are found to be temperature
dependent (Custodio 1993). We have previously reported the opposite effect of TAM
on membrane fluidity at low (1 mol%) and high (30 mol%) TAM concentrations
(Severcan et al. 2000). It will be very interesting to see how TAM affects membrane
fluidity at other concentrations, since dose dependent side effect of it was reported (De
Lima et al. 2003). Here we showed that TAM at low concentrations (1 and 3 mol%)
decreases dynamics. In contrast, at higher concentrations it increases the dynamics with
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more profound effect at concentrations of 18–24 mol%. This information is important
for the effective use of TAM in therapy and prevention of cancer. It was reported that
tumor cells might become resistant to the drugs. The reason for this phenomenon is still
unknown. One of the suggestions for that is the alteration in the dynamic properties of
the cell membrane (Ramu et al. 1983). Metastatic tumor cells have higher plasma
membrane fluidity than non-metastatic cells; thus, decreased membrane fluidity could
restore contact inhibition between the cells by increasing the rigidity of the cells (Ramu
et al.1983; Sok et al. 2002). In addition, cell division may be slower as a result of reduced
membrane fluidity (Ferreira et al. 2005).
TAM is proposed to be affective in premenopausal women at risk for breast cancer.
The previous studies showed that estrogen increases membrane fluidity on model (Boyar
and Severcan 1997) and brain membranes (Dicko et al. 1999). TAM can be affective due
to its membrane stabilizing effect at low concentrations and therefore may restore
membrane fludifying action of estrogen. (Dicko et al. 1999; Boyar and Severcan 1997).
It was previously reported that although the risk of breast cancer was reduced in the
TAM-treated group, several side effects such as deep vein thrombosis, pulmonary
emboli, increased incidence of stroke and cataract have also been observed (De Lima
et al. 2003). Therefore, it is very important to know precisely the concentration
dependent behavior of TAM on membrane fluidity. A recent study demonstrated that
the dose of TAM may be lowered without affecting its activity and reported that a lower
dose of TAM could reduce the side effects associated with treatment (De Lima et al.
2003; Decensi et al. 1998; Decensi et al. 1999), which support our hypothesis.
In conclusion, the results of the current study imply that, TAM may be beneficial in
therapy of and prevention from cancers because of its membrane stabilizing effect at
low concentrations.
Acknowledgements State Planning Organization of Turkey (DPT98K112530/AFP98010805) has
supported this work.
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