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Radiat. Phys. Chem. Vol. 49, No. 1, pp. 141-143, 1997 Copyright © 1997 Elsevier Science Ltd

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PHOTODYNAMIC ACTION OF MEROCYANINE 540 ON PLASMA MEMBRANE OF GLIOBLASTOMA CELLS

M R I N A L I N I SHARMA, PREETI G. JOSHI and N A N D A B. JOSHI Department of Biophysics, National Institute of Mental Health and Neuro Sciences, Bangalore

560 029, India

Abstract--Structural modifications in plasma membrane of human glioblastoma cells induced by photodynamic action of merocyanine 540 (MC540) were investigated using lipid and protein specific fluorescent probes. Photoirradiation of MC540 treated cells caused a decrease in the steady state anisotropy, decay time,limiting anisotropy and order parameter with an increase in the cone angle and rotational relaxation time of trimethylammoninm 1,6-diphenyl- 1,3,5-hexatriene (TMA-DPH). Increase m the lipid peroxidation, loss of SH groups, increase in the relaxation time of N-(1-Pyrene)-maleimide (PM) and cross linking of proteins were also observed on photosensitization of cells. These results suggest that photosensitization alters the structural organization of plasma membrane of glioblastoma cells. Copyright © 1997 Elsevier Science Ltd

INTRODUCTION

Photosensitizing action of MC540 on leukemia, lymphoma, neuroblastoma cells, enveloped viruses and virus infected cells has been exploited for autologous bone marrow purging and blood steriliza- tion (Sieber, 1987; Sieber et al., 1987, 1989). Recently, it has been shown that MC540 binds and photosensi- tizes glioblastoma cells also (Whelan et al., 1992; Sharma et al., 1994). Plasma membrane has been proposed as an important site of photodynamic action based on evidences such as photoinactivation of enzymes and photoperoxidation of membrane lipids (Gaffney and Sieber, 1990). Although photoin- duced changes in protein mobility and membrane fluidity have been reported in erythrocyte ghosts (Feix et al., 1991), structural alterations in plasma membrane have not been investigated in intact cells. In this communication, we report photoinduced modifications in the plasma membrane of glioblas- toma cells using lipid and protein specific probes.

MATERIALS AND METHODS

Chemicals and reagents

Merocyanine 540 (MC540), sodium pyruvate, and 2-thiobarbituric acid (TBA) were obtained from Sigma Chemical Co., St Louis (U.S.A.). Trimethy- lammonium 1,6-diphenyl-l,3,5-hexatriene (TMA- DPH) and N-(1-Pyrene)-maleimide (PM) were obtained from Molecular probes Inc., Eugene, OR (U.S.A.). Foetal calf serum (FCS) was procured from Northumbria Biological Ltd, Cramlington (U.K.). Eagle's minimum essential medium (EMEM) and phosphate buffered saline (PBS) obtained from Himedia, Bombay (India).

Methods

Human glioblastoma (U-87MG) cells obtained from American Type Culture Collection (ATCC) were grown at 37 ° in EMEM supplemented with 5% FCS, 5% bovine serum (BS), 1 mM sodium pyruvate and antibiotics. Cells were incubated with 15ttg/ml MC540 at 37 ° in EMEM containing 0.5% FCS and 0.5% BS for 1 h in dark. Cells were washed to remove extracellular dye and were released using 0.8% dispase and resuspended in PBS. MC540 treated cells were irradiated using two 40 W day light fluorescent tubes (Philips, India) with a fluence rate of 1 W/m 2 as measured by Kyoritsu Illuminometer model 5200 (Kyoritsu Electrical Instruments, Tokyo, Japan).

Cells (2 x 106/1111) were labeled with fluorescent probes, TMA-DPH (2 ttM) and PM (15 I~M) to study photo induced alterations in lipids and proteins respectively. The fluorescence spectra and steady state anisotropy measurements were made using SLM 8000C spectrofluorometer. The excitation wavelength was set at 337 nm, while emission was monitored at 415 and 396 nm for TMA-DPH and PM, respectively. Glan Thompson calcite prism polarizers were placed in the excitation and emission paths for measurement of anisotropy. For anisotropy measurements, samples were excited with vertically polarized light. Vertically (I~) and horizontally (Ivh) polarized fluorescence intensifies were measured and anisotropy (r) was calculated using the relationship (Lakowicz, 1983).

r = I,~ -- Glvh/Iw + 2GIvh (1)

Where G is the correction factor given by G = Ihv/Ihh, G factor is determined by measuring the fluorescence intensifies Ihv and In using horizontally polarized exciting light. MC540 treated cells with and without light irradiation but not labeled with fluorescent

141

142 Mrinalini Sharma e t al.

Table 1. Fluorescence anisotropy parameters of TMA-DPH incorporated cells. Cells were treated with 15 l.tg/ml of MC540 for 1 h in dark and irradiated for 60 rain. The values presented are mean _+ SD of four experiments

Treatment Limiting anisotropy (r~) Order parameter (S) Cone angle ( 0 ¢ ) Correlation time, ns(¢)

MC540 dark 0.225 _+ 0.005 0.757 ___ 0.009 34.09 ° __. 0.71 1.21 + 0.15 MC540 + light (60 min) "0.185 _+ 0.017 "0.686 +_ 0.033 "39.25 ° +_ 2.45 *4.46 + 0.75

*P < 0.0005, "P < 0.005, than dark controls.

p robes were used as b lanks for da rk and i r radia ted samples. Fluorescence intensifies I~, Lh, Ih~, and Ihh of b lanks were subt rac ted before calculat ing anisot ropy of T M A - D P H treated cells.

Decay t ime and t ime dependen t anisot ropy measurements were per formed using Ed inbu rgh C D 900 t ime resolved spectrofluorometer . Fluorescence intensi ty decay

I ( t ) = ~ e , e - ~/:' (2) i

was analyzed by non- l inear least square deconvolu- t ion procedure. Decay of vertically and hor izonta l ly polar ized fluorescence intensifies was measured and t ime dependen t anisot ropy was calculated using equa t ion (1). Limit ing anisot ropy (ro~) and ro ta t iona l corre la t ion t ime (¢ ) were ob ta ined from

r ( t ) = (ro - - r ~ ) e - ' / * i - - r~ . (3)

Cone angle (0¢) and order pa ramete r (S) were calculated f rom

r~ / ro = (S) 2 = [1/2(cos0~)(1 + cos0J] 2 . (4)

Lipid peroxidat ion was measured by th iobarbi tur ic acid assay (Atha r e t a l . , 1988).

RESULTS AND DISCUSSION

T M A - D P H is a lipid probe, which specifically labels p lasma membrane of in tac t cells. The fluorescence anisot ropy of T M A - D P H in MC540 treated cells in da rk was 0.259 + 0.007. The anisot ropy of cells i r radiated with light for 60 min was 0.220 ___ 0.006. The decrease in steady state an iso t ropy could be due to al terat ions in the mobil i ty of lipids or due to an increase in the decay time of f luorophore. In order to elucidate the change observed in steady state anisotropy, fluorescence life times of T M A - D P H in cells were measured. The decay curve was analyzed as described in the materials and method. The life times obta ined in MC540 treated cells were 3.02 + 0.16 and 8.05 + 0.29 ns with the fractional con t r ibu t ion of 0.28 ___ 0.07 and 0.7 + 0.07, respectively. On irradiat- ing with light for 60 min, life times decreased to 2.41 ___ 0.09 and 6.01 ___ 0.09 ns with fract ional contri- bu t ion of 0.36 + 0.03 and 0.64 + 0.03. The decrease

Z LIJ I-- Z

LU U Z ILl

" W r r O

. J tl_

OJ

F-

CE

1"0

0.5

5 360

\ \

I I [ 400 450 500 550

EMISSION WAVELENGTH ( nrn I

Fig. 1 Fluorescence emission spectra of PM labeled cells. Cells were incubated with 15 ~tg/ml of MC540 for 1 h in dark ( ) and photoirradiated with for 60 min (- - -). Cells were labeled with PM for 30 min

after photoirradiation.

Photodynamic action of MC540 on U87MG ceils 143

in the decay time observed in our study suggests change in the mobility of the probe on photosensi- tization of cells.

Time dependent anisotropy measurements of TMA-DPH were performed to further elucidate the dynamics of the membrane. Anisotropy decay curve generated from the vertically and horizontally polarized intensity decay was analyzed using equation (3) to determine the values of limiting anisotropy (r~) and rotational relaxation time (q~). Table 1 shows the effect of MC540 with and without photoirradiation on these parameters. The limiting anisotropy and order parameter (S) decreased, whereas the cone angle and correlation time increased on irradiating the cells treated with MC540. Decrease in the limiting anisotropy and order parameter reflects disorder in the plasma membrane of photosensitized cells. Increase in correlation time may be due to an increase in cone angle as time required for equilibrium in a larger angle will be more (Kawato et al., 1977).

The observed alterations in membrane may be due to the photoinduced lipid peroxidation. Lipid peroxidation quantitated by the amount of malondi- aldehyde in MC540 treated cells in dark was 0.0165 + 0.008 nmoles/106 cells. On irradiating the cells for 60 min malondialdehyde increased to 0.240 __+ 0.033 nmoles/106 cells. These results indicate that the peroxidation of membrane lipids could be responsible for increasing membrane fluidity and decrease in membrane order following photoirradia- tion. It has been reported earlier that oxidatively modified lipids alters lipid packing in the membrane leading to the change in the microenvironment of the probe (Wratten et al., 1992).

Photoinduced changes in the membrane protein SH groups were evaluated by labeling the cells with protein specific fluorescent probe PM. The fluor- escence spectra of PM in cells treated with MC540 in dark and photoirradiated for 60 rain are shown in Fig. 1. The spectra show monomer bands at 375, 396 and 415 nm and an excimer band at 470 nm. On photoirradiation, the total fluorescence intensity and the ratio of excimer to monomer intensity (470/396) decreased. The reduction in the total fluorescence intensity and excimer to monomer ratio of PM on irradiation suggests the cross linking of membrane proteins on photosensitization.

The mobility of proteins in MC540 treated cells was evaluated by measuring the anisotropy decay of PM in cells. The rotational relaxation time deter- mined using equation (3) was found to be 40.75 + 4.01 ns. On photoirradiation, it increased to 79.99 + 0.01 ns suggesting that photosensitization leads to a decrease in the mobility of proteins.

Increase in fluidity of membrane and decrease in protein mobility on photodynamic action of MC540 may be due to a change in the lipid-protein interaction in the cell membrane as suggested by Borochov and Shinitzky (1976). Proteins in the membrane are in equilibrium state due to their hydrophobic interaction with lipids and hydrophilic interaction with aqueous surroundings. Upon de- creasing order of lipid bilayer, lipid-protein inter- action will increase. As a result, protein may be squeezed into the hydrophobic core. This altered conformation may restrict the mobility of PM labeled proteins in the membrane.

Acknowledgements--This work was supported by Depart- ment of Science and Technology, Government of India, New Delhi.

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