Multidrug Resistance Reversal and Apoptosis Induction in Human

Article pubs.acs.org/jnp

Multidrug Resistance Reversal and Apoptosis Induction in Human Colon Cancer Cells by Some Flavonoids Present in Citrus Plants † ́ Olga Wesołowska,*,†,⊥ Jerzy Wiśniewski,†,⊥ Kamila Sroda-Pomianek, Aleksandra Bielawska-Pohl,‡ ‡ ‡ § Maria Paprocka, Danuta Duś, Noélia Duarte, Maria-José U. Ferreira,§ and Krystyna Michalak† †

Department of Biophysics, Wrocław Medical University, ul. Chałubińskiego 10, 50-368 Wrocław, Poland Laboratory of Glycobiology and Cellular Interactions, Department of Medical Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, ul. Weigla 12, 53-114 Wrocław, Poland § Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, Avenue Prof Gama Pinto, 1649.003, Lisbon, Portugal ‡

ABSTRACT: Multidrug resistance (MDR) of cancer cells constitutes one of the main reasons for chemotherapy failure. The search for nontoxic modulators that reduce MDR is a task of great importance. An ability to enhance apoptosis of resistant cells would also be beneficial. In the present study, the MDR reversal and apoptosis-inducing potency of three flavonoids produced by Citrus plants, namely, naringenin (1a), aromadendrin (2), and tangeretin (3), and the methylated naringenin derivatives (1b, 1c), have been studied in sensitive (LoVo) and multidrug-resistant (LoVo/Dx) human colon adenocarcinoma cells. Cytotoxicity of methoxylated flavonoids was higher as compared to hydroxylated analogues. Only 3 turned out to inhibit P-glycoprotein, as demonstrated by a rhodamine 123 accumulation assay. It also increased doxorubicin accumulation in LoVo/Dx cells and enabled doxorubicin to enter cellular nuclei. In addition, 3 was found to be an effective MDR modulator in resistant cells by sensitizing them to doxorubicin. Tangeretin-induced caspase-3 activation and elevated surface phosphatidylserine exposure demonstrated its apoptosis-inducing activity in LoVo/Dx cells, while the other flavonoids evaluated were not active. Additionally, 3 was more toxic to resistant rather than to sensitive cancer cells. Its apoptosis-inducing activity was also higher in LoVo/Dx than in LoVo cells. It was concluded that the activity of 3 against multidrug-resistant cancer cells may be enhanced by its apoptosis-inducing activity. activities,8 to inhibit aromatase,9 to influence gene transcription mediated by the aryl hydrocarbon receptor,10 and to be an effective inhibitor of multidrug transporters from the MRP family.11,12 Aromadendrin (2), (+)-dihydrokaempferol, apart from being present in other plants, has also been found to be a component of the molasses of tangerine orange.13 It has been identified as a weak inhibitor of monoamine oxidase14 and also of the multidrug transporter MRP1.12 Tangeretin (3) is an example of a polymethoxylated flavone found abundantly in citrus fruit peel.15 This compound was demonstrated to be an antiproliferative agent against several types of cancer cell lines,16 to induce G1 cell-cycle arrest in human colorectal carcinoma,17 and to regulate gene transcription.18 In human intestinal cells, tangeretin activates the pregnane X receptor (PXR), which constitutes a key regulator for the expression of genes encoding drug-metabolizing enzymes (e.g., cytochrome P450) and transporters (e.g., P-glycoprotein).18 Recently, 3 has also been shown to inhibit P-glycoprotein and to reverse multidrug resistance in adriamycin-resistant myelogenous leukemia cells.19

Citrus fruits, fresh or processed (e.g., juices), are consumed popularly all over the world. According to a UNCTAD report,1 the total annual citrus fruit production exceeded 105 million tons in the period 2000−2004 and was expected to grow continuously. Almost half of the world citrus market constitutes oranges, while lemons, limes, grapefruits, pomelos, tangerines, and mandarins are produced in lower quantities. Citrus species are rich in bioactive compounds, including flavonoids. Flavonoids are polyphenolic compounds for which numerous biological activities have been reported, including antioxidative, anti-inflammatory, and antimicrobial properties as well as the potential ability to prevent cancer and cardiovascular diseases and to reduce the symptoms of menopause.2,3 Numerous flavonoids (mostly flavanone and flavone glycosides as well as many polymethoxyflavones) have been identified in citrus fruits, juices, and peels.4 Recent research has concentrated mainly on their anti-inflammatory and enzyme inhibitory properties, which have been suggested to be at least partially responsible for the possible protective effect of Citrus flavonoids against cardiovascular diseases and some types of cancer.5 The naringenin glycoside, naringin, is the most abundant flavonoid in grapefruits and is responsible for the bitter taste.6 Naringenin (1a) was shown to possess antibacterial7 and anti-inflammatory © 2012 American Chemical Society and American Society of Pharmacognosy

Received: May 15, 2012 Published: November 8, 2012 1896

dx.doi.org/10.1021/np3003468 | J. Nat. Prod. 2012, 75, 1896−1902

Journal of Natural Products

Article

The multidrug resistance (MDR) of cancer cells results in their simultaneous resistance to many chemotherapeutic drugs that are structurally and functionally unrelated. MDR may be an intrinsic feature of a given cancer type or may be acquired during the course of initial chemotherapy. Currently, MDR represents one of the main reasons of chemotherapy failure. Multidrug resistance may be associated with numerous changes in the functioning of cancer cells. The overexpression of ATPbinding cassette transporters (ABC transporters) of wide substrate specificity as well as the changes in apoptotic pathways (e.g., resistance to pro-apoptotic signals) are listed typically among the most important molecular mechanisms associated with MDR.20 MDR-engaged ABC transporters are transmembrane proteins that utilize the energy of ATP hydrolysis to pump anticancer drugs out of a cell, thereby reducing their intracellular concentration. The majority of clinically significant cases of MDR seem to be the result of overexpression of three ABC transporters characterized by broad substrate specificity: P-glycoprotein (P-gp, ABCB1), multidrug resistance-associated protein 1 (MRP1, ABCC1), and breast cancer resistance protein (BCRP, ABCG2).21 Additionally, the sensitivity of drug-resistant cancer cells to pro-apoptotic signals is often impaired. 22 Therefore, a continuing search for compounds able to reduce multidrug resistance (MDR modulators) remains a task of great importance. Such modulators should be nontoxic themselves and should not interfere with any other cellular processes. However, a concomitant ability to enhance apoptosis of resistant cancer cells is beneficial. In the present study, the activity was determined of several flavonoids previously identified from Citrus plant products, viz., naringenin (1a), aromadendrin (2), and tangeretin (3), as well as the methylated naringenin derivatives naringenin 7-methyl ether (1b) and naringenin 7,4′-dimethyl ether (1c), to reverse multidrug resistance and to induce apoptosis in human colon adenocarcinoma cells. A chemotherapy-sensitive cell line (LoVo) and its multidrug-resistant subline (LoVo/Dx) were used. These cell lines have been characterized previously.23 Both express several ABC transporters (P-gp, MRP1, BCRP), and it was demonstrated that the main difference between the sensitive and the resistant cells lies in the overexpression of Pglycoprotein in LoVo/Dx cells. The ability of flavonoids to influence rhodamine 123 accumulation, to affect intracellular doxorubicin localization, and to revert doxorubicin sensitivity in resistant cells as well as their ability to induce apoptosis (detected via phosphatidylserine exposure on the cell surface and the stimulation of caspase-3 activity) was investigated. Tangeretin (3) was found to be the most active multidrug resistance modulator and apoptosis inducer among the compounds studied.

Figure 1. Cytotoxicity of naringenin (1a, full circles), aromadendrin (2, empty circles), naringenin 7-methyl ether (1b, triangles), naringenin 7,4′-dimethyl ether (1c, full squares), and tangeretin (3, empty squares) to LoVo (A) and Lovo/Dx (B) cells. Means ± SD of three experiments are presented.

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RESULTS AND DISCUSSION The cytotoxicity of all compounds to chemotherapy-sensitive LoVo cells and to multidrug-resistant LoVo/Dx cells was evaluated (Figure 1). Both in LoVo and LoVo/Dx cells, the cytotoxicity of the flavonoids studied increased in the order 2 < 1a < 1c < 1b < 3. Most of the flavonoids studied were more active against the drug-sensitive cell line, and only 1b displayed similar cytotoxicity to both cell lines, with 3 significantly more cytotoxic to resistant LoVo/Dx cells than to their sensitive counterparts. A higher cytotoxicity of 3 to resistant than to sensitive cells has been reported previously for MOLT-4 human T lymphoblastoid leukemia cells.24

Generally, flavonoids with no methoxy groups were found to be less cytotoxic to human colon cancer cells as compared to the derivatives possessing one or two methoxy groups, while the polymethoxylated flavone 3 was the most cytotoxic compound evaluated. The higher cytotoxicity of methylated flavones to cancer cells as compared to their unmethylated counterparts has been reported previously.16,25,26 Naringenin (1a) and aromadendin (2) were the least cytotoxic compounds, especially to the resistant cells used. This is in agreement with the previous literature, since IC50 values published for these two flavonoids range from 100 to several hundred μM depending on the cancer cell line used.27,28 In the case of tangeretin (3) 1897



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sensitive cancer cells. In resistant cells, only a weak fluorescent signal was recorded and doxorubicin remained excluded from cellular nuclei. The addition of 3 had no influence on doxorubicin accumulation and localization in LoVo cells (Figure 3C), but it had a dramatic effect in LoVo/Dx cells (Figure 3D). The intensity of the fluorescent signal recorded in resistant cancer cells increased. Doxorubicin intracellular localization was also affected; in the presence of 3 the drug was able to enter the nuclei of LoVo/Dx cells. Finally, the ability of 3 to sensitize the resistant cells to doxorubicin was investigated. As expected, doxorubicin was found to be more cytotoxic to sensitive cancer cells (Figure 4A) than to resistant cells (Figure 4B). The treatment of the cells with 5 μM 3 did not change doxorubicin cytotoxicity to LoVo cells (Figure 4A), but it did result in an increase of the cytotoxicity to LoVo/Dx cells (Figure 4B). Within the set of the compounds investigated, only 3 turned out to be a P-gp inhibitor. Its potency in increasing rhodamine 123 accumulation in LoVo/Dx cells was, however, lower than the potency of the well-known P-gp inhibitor verapamil, measured in the same cellular model.23 Tangeretin (3) was also demonstrated to increase doxorubicin accumulation in LoVo/ Dx cells and to enable doxorubicin to enter cellular nuclei. Nuclei are a cellular target of this anticancer drug, and doxorubicin is thought to exert its antineoplastic effect by intercalating DNA and inhibiting its biosynthesis.31 Finally, 3 was shown to be an effective MDR modulator in resistant colon carcinoma cells able to increase their sensitivity to doxorubicin. Tangeretin (3) was previously identified as a P-gp inhibitor in doxorubicin-resistant K562/ADM human myelogenous leukemia cells,32 daunorubicin-resistant MOLT-4/DNR T-lymphoblastoid leukemia cells,24 and the Caco-2 cell monolayer model.33 Naringenin (1a), in turn, was demonstrated as an inhibitor of several ABC transporters including MRP1,11,12 MRP4,11 and BCRP,34 but contradictory reports are evident dealing with its interaction with P-gp. Depending on the cellular model used, 1a was reported to be a P-gp inhibitor,35 to have no effect on this transporter,36 and even to stimulate its transport activity.37 All flavonoids studied were tested for their ability to induce apoptosis in human colon cancer cells. The exposure of phosphatidylserine on the membrane surface has been quantified by means of flow cytometry, and the results are presented in Table 1. Thus, all compounds at 100 μM slightly increased the number of apoptotic cells in the LoVo cell line. However, none of the results obtained were statistically significant, with the exception of camptothecin, an apoptosis inducer used as a positive control. On the other hand, only camptothecin and tangeretin (3) were able to increase the percentage of apoptotic cells in the population of LoVo/Dx cells. All the flavonoids, with the exception of 1a and 2, induced also some degree of necrosis in both LoVo and LoVo/Dx cells. Generally, the more cytotoxic compounds induced more necrosis in human adenocaricinoma cells. It is worth mentioning that the percentage of camptothecin-induced apoptosis was similar in both cell lines, whereas treatment with 3 at all concentrations tested resulted in a higher number of apoptotic cells identified within the population of LoVo/Dx cells as opposed to LoVo cells. The comparison of equitoxic concentrations of 3 (25 and 75 μM were chosen as approximate IC50 values for LoVo/Dx and LoVo cells, respectively) suggested that this compound exhibits higher apoptosisinducing activity in the multidrug-resistant cells.

IC50 values reported in the literature typically aggregate between 20 and 40 μM.17,29,30 In order to identify putative MDR modulators among the flavonoids investigated, a flow cytometry test based on the measurement of intracellular rhodamine 123 (fluorescent substrate analogue for P-gp) accumulation was applied. Although several MDR-associated transporters are expressed in multidrug-resistant colon cancer cells (LoVo/Dx), this assay has been shown previously to be indicative of P-gp transport activity.23 The mean fluorescence of the cell population (±SD of three experiments) was more than 8 times higher in LoVo cells (703 ± 122) as compared to LoVo/Dx cells (83 ± 23). Among the group of flavonoids studied, all compounds showed no ability to inhibit P-gp (data not shown), with the exception of tangeretin (3). Sensitive LoVo cells express P-gp, but at significantly lower level than LoVo/Dx cells,24 so therefore the rise in rhodamine 123 accumulation induced by 3 was visible in both sensitive and resistant cells (Figure 2A). The P-gp inhibition effect was much more pronounced in LoVo/Dx cells, as demonstrated by FIR values for tangeretin (Figure 2B).

Figure 2. Influence of tangeretin (3) on rhodamine 123 accumulation in cancer cells. Fluorescence of LoVo (triangles) and LoVo/Dx (squares) cell populations is presented (A) as well as FIR values (B). Means ± SD of three experiments are presented. Only FIR values obtained for 75 and 100 μM of tangeretin were found to be statistically significant (p < 0.05) as compared to the control.

Tangeretin (3) was tested also for its ability to influence the intracellular localization of doxorubicin. Doxorubicin displays strong intrinsic fluorescence, so its intracellular localization can be visualized with the use of fluorescence microscopy. Images of LoVo and LoVo/Dx cells treated with doxorubicin alone are presented in Figure 3A and B, respectively. It can be noted that LoVo cells accumulated more doxorubicin than LoVo/Dx cells and that the drug localized predominantly in the nuclei of 1898



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Figure 3. Doxorubicin accumulation in LoVo (A) and LoVo/Dx cells (B) treated with 100 μM tangeretin (3) (C and D for LoVo and LoVo/Dx, respectively). Doxorubicin concentration was 50 μM. Scale bar is 50 μm. Illumination conditions were the same for all images.

in the present study is noteworthy and points to the involvement of some factors specific for the resistant subline in the process of apoptosis induced by this flavonoid. An analysis of the literature reveals that the potency of 3 in inducing apoptosis clearly depends on the particular cell line being investigated. Tangeretin (3) was reported previously to induce apoptosis in K562 human erythroleukemia cells,30 SHSY5Y human neuroblastoma cells,42 and HL-60 human promyelocytic leukemia cells43 but not in MOLT-4 Tlymphocytic leukemia cells,30,43 COLO 20517 and HT-29 colorectal carcinoma cells,29 and MDA-MB-435 and MCF-7 breast cancer cells.29 Tangeretin-induced cell-cycle G1 arrest was, however, reported in cancer cell lines in which tangeretin did not induce apoptosis.17,24,29 The decrease of mitochondrial membrane potential,42 activation of caspases,30 and DNA fragmentation30 were identified as the criteria of tangeretininduced apoptosis attributed to the activation of the intrinsic apoptosis pathway by this flavonoid. Naringenin (1a) is reported to be an effective apoptosis inducer in several cancer cell lines, including HL-60 human promyelocytic leukemia cells,44 THP-1 human myeloid leukemia cells,45 and Caco-2 colon cancer cells,27 in contrast to the present findings in sensitive and resistant colon cancer cells (LoVo and LoVo/Dx). Similarly, as for 3, the mechanism of naringenin-induced apoptosis also included the decrease of mitochondrial potential45 and caspase activation,44,45 which might suggest the activation of the same apoptosis pathways by both flavonoids. In conclusion, within a set of flavonoids produced by Citrus plants studied, tangeretin (3) was identified as an effective MDR modulator and an apoptosis inducer in LoVo/Dx multidrug-resistant colon cancer cells. Its modulatory activity

The ability of 3 to activate one of the apoptosis-involved proteases, caspase-3, was also investigated (Table 2). The camptothecin-induced increase of caspase 3 activity was statistically significant (p < 0.05) in both LoVo and LoVo/Dx cells, whereas for 3 only the results obtained in LoVo/Dx cells turned out to be significant. In a similar manner to superficial phosphatidylserine exposure, 3 increased caspase 3 activity to a higher extent in the resistant than in the sensitive adenocarcinoma cells. On the other hand, equitoxic concentrations of 3 caused similar changes in caspase 3 activity in both types of cells. Among the group of flavonoids tested, 3 was the only apoptosis inducer in colon cancer cells. The effect of 3 was, however, statistically significant only in the multidrug-resistant LoVo/Dx cells. Tangeretin (3) increased the number of apoptotic cells within the population of LoVo/Dx cells as judged by flow cytometric quantification of cells that expose phosphatidylserine on their surfaces but that have unimpaired membrane integrity. The loss of membrane asymmetry occurs at the beginning of the execution phase of apoptosis and is believed to last until the end of this phase.38 Moreover, 3 was shown to activate caspase-3 in LoVo/Dx cells. Caspase-3, an aspartate-specific cysteine protease, is one of the key executioners of apoptosis, responsible for proteolytic cleavage of many cellular proteins.39 It is activated during the early stage of the execution phase via cytochrome-dependent proteolytic processing by caspase-9, which occurs after the release of cytochrome c from disrupted mitochondria40 or via cleavage by caspase 8 activated in the complex of Fas-associated death domain (FADD) with one of the death receptors.41 The ability of 3 to induce apoptosis in multidrug-resistant LoVo/Dx cells to a much higher extent than in sensitive LoVo cells observed 1899



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Table 2. Effect of Tangeretin (3) on Relative Caspase 3 Activity in LoVo/Dx and LoVo Cellsa caspase 3 activity compound

concentration [μM]

control camptothecin tangeretin (3)

LoVo 1 3.40 1.04 2.06 2.17

15 25 75 100

± ± ± ±

0.29* 0.21 0.26 0.29

LoVo/Dx 1 3.83 2.08 2.37 2.71

± ± ± ±

0.31* 0.20* 0.31* 0.28*

a

Calculated as a ratio of the amount of cleaved caspase 3 substrate DEVD-pNA in a sample treated with an inductor for 48 h to the amount in a control sample treated with medium only. Means ± SD of three experiments are presented. Results marked with an asterisk are significant statistically (p < 0.05).

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EXPERIMENTAL SECTION

Materials. Naringenin (1a) and aromadendrin (2) are flavonoids present in Citrus plants, but the samples of these compounds used in the present study were isolated from a methanol extract of Euphorbia tuckeyana Steud. (Euphorbiaceae) aerial parts (procedure described in ref 28). The derivatives naringenin 7-methyl ether (1b) and naringenin 7,4′-dimethyl ether (1c) were obtained by methylation of 1a by the method described previously in detail.46 The purity of the isolated compounds and derivatives was more than 95% (determined by analytical HPLC). Tangeretin (3) (≥95%, HPLC), doxorubicin, rhodamine 123, camptothecin, propidium iodide, and sulforhodamine B were purchased from Sigma (Poznan, Poland). Annexin-V, AlexaFluor 488 conjugate, was obtained from Molecular Probes (Eugene, OR, USA). Other reagents used were of analytical grade. Rhodamine 123 and doxorubicin were dissolved in water. All other compounds were dissolved in dimethyl sulfoxide (DMSO). Cells. A human colorectal adenocarcinoma cell line, LoVo, and its resistant subline, LoVo/Dx, obtained by prolonged exposure to doxorubicin,47 were used. Cultivation conditions were Ham’s F12 medium (with the addition of 10% fetal bovine serum, L-glutamine, antibiotics, and, in case of LoVo/Dx cells, doxorubicin at 100 ng/mL), 37 °C, and 5% CO2. Cytotoxicity Assay. Cells were seeded (30 000 cells/well) onto 96-well plates in 75 μL of medium and allowed to attach (60 min, 37 °C). Next, an equal volume of medium containing the studied compound was added, and the incubation was continued for 72 h. The sulforhodamine B (SRB) assay was performed according to the method described previously.48 Absorbance at 492 nm was recorded, and the percentage of cell survival was calculated as (A492 of treated cells/A492 of control cells) × 100%. The control cells were treated with medium only. Cytotoxicity of DMSO (maximal concentration in

Figure 4. Influence of tangeretin (3) on doxorubicin cytotoxicity in LoVo (A) and LoVo/Dx (B) cells. The circles represent doxorubicin alone, and the squares doxorubicin plus 5 μM tangeretin. Means ± SD of three experiments are presented. The differences between samples treated with doxorubicin alone vs doxorubicin plus tangeretin were statistically significant (p < 0.05) only for LoVo/Dx cells (apart from the result obtained at 8.6 μM doxorubicin).

was manifested in an ability to inhibit P-glycoprotein, to affect intracellular localization of doxorubicin, and to sensitize resistant cells to this anticancer drug. The ability of 3 to induce apoptosis in a resistant cell line but not in its sensitive counterpart is of particular interest. The activity of 3 against multidrug-resistant cancer cells might be therefore enhanced by its apoptosis-inducing activity. The precise explanation of tangeretin’s mechanism of action in resistant cells would, however, require further studies with a higher number of sensitive and drug-resistant cell lines involved.

Table 1. Effect of Flavonoids on Apoptosis Induction in LoVo/Dx and LoVo Cellsa LoVo compound +

concentration [μM]

+

annexin-V PI camptothecin naringenin (1a) aromadendrin (2) naringenin 7-methyl ether (1b) naringenin 7,4′-dimethyl ether (1c) tangeretin (3)

15 100 100 100 100 25 75 100

apoptosis [%] 6.68 17.61 15.44 10.96 18.16 14.10 11.57 12.83 15.19

± ± ± ± ± ± ± ± ±

1.77 4.41* 8.93 4.49 10.5 6.61 1.63 2.89 3.91

LoVo/Dx necrosis [%] 10.44 23.81 8.16 10.02 25.28 17.82 25.82 29.61 23.75

± ± ± ± ± ± ± ± ±

3.23 7.86 0.29 2.25 8.18 2.59 0.51 8.19 3.22

apoptosis [%] 6.16 17.56 6.98 9.38 4.87 9.67 13.87 13.35 23.05

± ± ± ± ± ± ± ± ±

1.67 1.75* 2.51 1.92 2.03 6.92 2.59* 1.62* 2.28*

necrosis [%] 9.57 17.96 5.65 6.83 18.89 17.74 17.55 17.45 21.75

± ± ± ± ± ± ± ± ±

2.83 2.26 0.67 1.80 3.42 0.81 4.26 0.62 0.28

a

Cells were recognized as viable (annexin-V and PI negative), apoptotic (annexin-V positive and PI negative), and necrotic (annexin-V and PI positive) based on the measurement of cell-associated fluorescence of annexin-V AlexaFluor conjugate and PI. Means ± SD of three experiments are presented. Results marked with an asterisk are significant statistically (p < 0.05). 1900



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samples 0.3%) on the cells was found to be negligible (100 ± 5% survived experiments). Accumulation of Rhodamine 123. Cells (300 000 cells/mL in serum- and phenol red-free medium) were incubated with the appropriate concentration (5−100 μM) of the modulator (15 min, room temperature). Then, rhodamine 123 was added (final concentration 2 μM), and the incubation was continued for 60 min at 37 °C. After washing of the cells and resuspension in PBS the fluorescence of the cell population was analyzed by flow cytometry (Becton Dickinson FACSCalibur with a 488 nm argon laser). Fluorescence was recorded via a 530/30 nm band-pass filter. For each experiment 5000 events were recorded and analyzed with the use of Cell Quest software (Becton Dickinson). The presence of DMSO (maximal concentration in samples 0.8%) changed the fluorescence of the cell population by less than 5% as compared to the cell population untreated with this solvent. Fluorescence intensity ratio (FIR) was calculated on the basis of measured fluorescence values (FL):

FIR =

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AUTHOR INFORMATION

Corresponding Author

*Tel: +48-71-7841415. Fax: +48-71-7848800. E-mail: olga. [email protected]. Author Contributions ⊥

These authors contributed equally to this work

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financed by Polish Ministry of Science and Higher Education funds for Wroclaw Medical University and also by Portuguese Foundation for Science and Technology (FCT, project PTDC/QUI-QUI/099815/2008).

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(FL LoVoDx treated)/(FL LoVoDx control) (FL LoVo treated)/(FL LoVo control)

REFERENCES

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Control samples were treated with medium containing 2 μM rhodamine 123 (no modulator). Fluorescence Microscopy. The cells seeded onto an eight-well μslide microscopy chamber (Ibidi, Munich, Germany) at 15 000 cells/ well were cultivated for 48 h. Then, a fresh portion of F12 medium was added containing 50 μM doxorubicin (plus 100 μM modulator in treated samples), and cells were incubated for 60 min at 37 °C. Cells were washed twice with PBS and once with serum- and phenol redfree medium. Images were collected with a Nikon Eclipse TE2000-E microscope, equipped with a PlanFluor 40× (0.60) objective. A Nikon G-2E/C bandpass filter was used (excitation passband 528−553 nm, emission window 578−633 nm). Apoptosis Induction Assays. For detection of phosphatidylserine exposure on the surface of apoptotic cells flow cytometry was applied. Cells were grown for 24 h in six-well plates (700 000 cells/ well). Next, the modulator was added (final concentration was 100 μM), and the cells were cultivated for a further 48 h. For analysis, 100 000 cells were collected, washed, and incubated with annexin-V, AlexaFluor conjugate (15 min, room temperature, buffer: 10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Propidium iodide (at 7.5 nM) was added to the cells 2 min before measurement. The fluorescence of the cell population was measured by a Becton Dickinson FACSCalibur instrument (488 nm argon laser). Fluorescence was recorded with a 530/30 nm bandpass filter for AlexaFluor 488 and 585/42 nm bandpass filter for propidium iodide (PI). A total of 5000 events were registered and subsequently analyzed by quadrant statistics with the use of Cell Quest software (Becton Dickinson) for the presence of viable (annexin-V and PI negative), apoptotic (annexin-V positive and PI negative), and necrotic (annexinV and PI positive) cells. Camptothecin was used as a positive control. Evaluation of caspase-3 activation was performed using a commercially available kit (GenScript, Piscataway, NJ, USA) according to the manufacturer’s protocol. In brief, cells were seeded (800 000/ well) onto a six-well plate in 2 mL of medium and allowed to attach for 24 h. Next, the test compound at the appropriate concentration was added in a fresh portion of medium, and the cells were further incubated for 48 h. At the end of the incubation, cells were scraped and centrifuged (2000g, 5 min). Caspase-3 activity detection is based on spectrophotometric detection (A405) of the chromophore pnitroanilide (pNA) released by the enzyme by proteolytic cleavage of its substrate DEVD-pNA. The relative increase of caspase-3 activity was determined by calculating the ratio of the absorbance of pNA in a studied sample (treated with an inductor) to a control. Statistical Analysis. All experiments were performed in triplicate. Data are described as means ± SD and analyzed by the Student’s t test. p-Values below 0.05 were considered to be statistically significant. 1901



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Multidrug Resistance Reversal and Apoptosis Induction in Human

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