ANTICANCER RESEARCH 33: 857-864 (2013) The Influence of Phenothiazine Derivatives on Intracellular Accumulation of Cationic Cyanine Dye DiOC6(3) in LoVo-DX Cells ANDRZEJ POŁA1, DANIELA MOSIĄDZ1, JOLANTA SACZKO2, TERESA MODRZYCKA1 and KRYSTYNA MICHALAK1 Departments of 1Biophysics and 2Medical Biochemistry, Wrocław Medical University, Wroclaw, Poland Abstract. Aim: This study aimed to evaluate the influence phenomenon of human cancer cells, which is manifested by of phenothiazine derivatives (PDs) on the intracellular transport of a broad array of chemical compounds out of accumulation of cyanine dye DiOC6(3) in doxorubicin- cells (6, 7). Among MDR transporters, the earliest discovered resistant LoVo-DX cell line, with overexpression of P- was P-gp (8), hence, the molecular mechanism of its action glycoprotein. Materials and Methods: In order to maintain is best-understood. Most researchers suggest that P-gp acts a high expression level of P-gp, the LoVo-DX cells were as a pump that removes cytotoxic drugs, as well as other grown in the presence of doxorubicin (100 ng/ml). The time- structurally- and functionally-unrelated compounds, trying to dependent fluorescence signal (T-DFS) of the intracellular penetrate the cancer cell interior (4, 9-16). It seems that the accumulation of DiOC6(3), in the presence of PDs, was then electrical charge of transported molecules plays an important recorded. The rate constants k1, k2, k3 and amplitudes of T- role in recognizing compounds as substrates for MDR DFS, describing the intracellular accumulation process, transporters. P-gp has very high affinity for organic cations, were determined based on the respective theoretical whereas ABCC1 substrates share the property of being equation. Results: The values of k1 and k2 were dependent amphiphilic organic anions and glutathione or glucuronate on the hydrophobicity (logP) of the PDs used as drug conjugates (17, 18). It was found that amphiphilic drugs (19) resistance modulators. A rise of k1 and k2 values was and anions (20, 21), as well as cations (22, 23), can be observed when the logP of PDs increased. Conclusion: We transported by ABCG2. suggest that the k1 and k2 rate constants could be regarded Higher intracellular accumulation of anticancer drugs was as useful parameters for assessment of PDs as well as of observed in cell lines with low P-gp expression, as compared other compounds of potential application as reversers of to their resistant counterparts with P-gp overexpression (11, multidrug resistance. 24). Similarly, a lower intracellular concentration of cationic cyanine dyes was observed in cancer cells with high Multidrug resistance (MDR) of cancer cells is a basic reason expression of this transporter (25-27). Spectral properties of for frequent failure of chemotherapy in cancer treatment. The cyanines are very useful to determine the kinetic parameters molecular mechanism of MDR is multifactorial (1-3) but the describing the process of intracellular dye accumulation. The classical MDR phenotype is associated with overexpression kinetic parameters may be estimated from the model proposed of multidrug transporters belonging to the superfamily of by Wadkins and Houghton (26), which was established on the ATP-binding cassette proteins (ABC proteins), in the plasma basis of analyses of time-dependent fluorescence signals (T- membrane of cancer cells (4, 5). From the superfamily of DFS) during intracellular accumulation of cyanine dye in ABC proteins, mainly P-glycoprotein (P-gp; ABCB1), cancer cells with different P-gp expression levels. ABCC1 and ABCG2 are responsible for the MDR Many different chemical compounds can reverse or modulate MDR in cancer cells (5, 28). A very important group of compounds which in in vitro experiments has been shown to be MDR-reversing agents, are phenothiazine Correspondence to: Andrzej Poła, Department of Biophysics, derivatives (PDs). Phenothiazines are used in the treatment Wrocław Medical University, ul. Chałubińskiego 9, 50-368 Wrocław, Poland. E-mail: [email protected] of psychiatric disorders, and PDs are well-known substrates of P-gp (29-32). Key Words: LoVo, LoVo/DX, cyanine dyes, intracellular In the present article, we examined the influence of PDs, accumulation, phenothiazines. belonging to the perazine and promazine group, on the 0250-7005/2013 $2.00+.40 857 ANTICANCER RESEARCH 33: 857-864 (2013) intracellular accumulation of cationic carbocyanine dye Table I. Chemical structures of cyanine dye DiOC6(3) and phenothiazine derivatives from the promazine and perazine groups, and [DiOC6(3)] in colonic cancer cells with P-gp overexpression. Since both DiOC (3) and PDs are P-gp substrates, we assumed calculated values of logP for these compounds. *Calculated using the 6 software: SPARTAN ’08. that the presence of PDs in the cell suspension might affect the process of cyanine dye intracellular accumulation and, in consequence, changes in kinetic parameters describing this process should be detected. In the interpretation the results of our experiments a potential influence of ABCC1 and ABCG2 on intracellular accumulation of DiOC6(3) was excluded. The cationic dye used in our study was not a substrate in the case of ABCC1 protein. On the other hand, the expression ABCG2 protein was at the same level for both the LoVo and the LoVo-DX cell lines, what was confirmed in our laboratory (33). Materials and Methods Reagents. Promazine, chlorpromazine, trifluopromazine, prochlorpromazine, dimethyl sulfoxide (DMSO), DiOC6(3), dioxane, chloroform, butanol, acetone, ethanol, methanol, Dulbecco’s modified Eagle’s medium F12 Ham, cell dissociation solution non-enzymatic, doxorubicin hydrochloride, antibiotic antimycotic solution and fluorescein 5(6)-isothiocyanate (FITC) were purchased from Sigma, Poznań, Poland. Fetal bovine serum (FBS) was purchased from Gibco, Warsaw, Poland, L(+)-glutamine from BDH Prolabo, Wrocław, Poland and trifluoperazine from ICN Biomedicals Inc., Gdańsk, Poland. Perazine was supplied by FSP Galena, Wrocław, Poland. The antibody for P-gp was purchased from Santa Crus Biotechnology, Dallas, Texas, USA. Stock solutions of PDs (5 mM) and DiOC6(3) (400 μM) were prepared in DMSO. The chemical structure, kind of substituent at position 2 of the phenothiazine ring and calculated values of partition co-efficient (logP) for PDs used in these studies, are presented in Table I. Intracellular dye accumulation. The emission intensity of T-DFS Cell preparation. Doxorubicin-sensitive LoVo and doxorubicin- during intracellular accumulation of the dye was monitored at resistant LoVo-DX cells were obtained from Institute of λem=509 nm with excitation wavelength λex=482 nm. Fluorescence Immunology and Experimental Therapy (Polish Academy of measurements were carried-out with a Perkin-Elmer LS-50B Sciences, Wroclaw, Poland). Both cell lines were grown in spectrofluorimeter. A quartz cuvette, containing DiOC6(3) solution Dulbecco’s medium, supplemented with 10% FBS, L-glutamine and in cell medium (without FBS, glutamine, antibiotics) was placed in antibiotic solution. In order to maintain a high level expression of P- the spectrofluorimeter and then an appropriate volume of cell gp, the LoVo-DX cells were grown in the presence of doxorubicin suspension was added (sample was continuously stirred). The final (100 ng/ml). Cells were cultured at 37˚C in a humidified atmosphere concentration of DiOC6(3) was 0.4 μM and the cell density was 5 3 of 5% CO2. In log-phase growth, they were removed from the flask 2.5×10 cells/cm . To measure the kinetics of dye accumulation in surface with non-enzymatic cell dissociation solution. The density the presence of PD, an appropriate amount of PD in solution was of cells in suspension was determined using a Bürker mixed with the dye solution and then the cell suspension was added. haemocytometer and was usually about 106/cm3. The final concentration of PDs was 15 μM. Determination of P-gp expression by immunofluorescence. Cells in Mathematical analysis. The mathematical analysis of the intracellular growth medium were plated on cover glasses and were grown for accumulation of carbocyanine dyes was given by Wadkins and one or two days. They were fixed in 4% formalin buffer and washed Houghton (26). In our study, an equation (Eqtn 1) was applied to with PBS. The intracellular localization of P-gp was determined with estimate parameters characterizing dye accumulation in cells. The antibody detector labeled with FITC. Samples were attached to observed fluorescence intensity F(t) (T-DFS) for cyanine dye in the microscope slides using fluorescence mounting medium. form of monomers during its accumulation in cancer cells is given by: Fluorescence was monitored using a confocal scanning laser microscope (Carl Zeiss GmbH, Jena, Germany) with excitation wavelength λex of 470±20 nm and fluorescence emission recorded at λem=525±20 nm. (Eqtn 1) 858 Poła et al: Effect of PDs on DiOC6(3) Uptake in LoVo-DX Cells Results Confocal microscopy studies. Using confocal microscopy, we determined the expression of transporting membrane protein, P-gp, in both cell lines. We observed a significantly higher expression of P-gp in plasma and mitochondrial membranes of LoVo-DX cells, as compared to LoVo cells (Figure 1). Intracellular accumulation of DiOC6(3) in LoVo-DX and LoVo cells determined by fluorescence spectroscopy. The changes of T-DFS during intracellular accumulation of 5 3 DiOC6(3) for both cell lines (2.5×10 /cm ) are shown in Figure 2. The estimated value of rate constants k1 and k2 were higher for LoVo cells than for LoVo-DX cells. Like the rate constants the amplitudes A1 and A2 were also higher for LoVo cells (Table II). The solid lines in Figure 2 represent the theoretically calculated curves obtained by fitting of experimental data to Equation 1. Accumulation of DiOC6(3) in LoVo-DX cells in the presence of PDs determined by fluorescence spectroscopy. The addition of a given PD to the DiOC6(3) buffer solution (without the cells) caused a slight (less than 1%) decrease in the amplitude of T-DFS for the dye monomer (data not shown).