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Modulation of Multidrug Resistance and Apoptosis of Cancer Cells by Selected Carotenoids

JOSEPH MOLNÁR1, NÓRA GYÉMÁNT1, ILONA MUCSI1, ANNAMÁRIA MOLNÁR1, MARGARET SZABó2, TAMÁS KÖRTVÉLYESI3, ANDRÁS VARGA4, PÉTER MOLNÁR5 and GYULA TÓTH5

1Institute of Medical Microbiology and Immunology, 2Department of Plant Physiology and 3Department of Physical Chemistry, University of Szeged, Hungary; 4Department of Molecular Parasitology, Humboldt University, Berlin, Germany, 5Department of Biochemistry and Medical Chemistry, University of Pécs, Hungary

Abstract. The multidrug resistance (MDR) proteins that after this finding, rutin, as vitamin P, was also extracted belong to the ATP-binding casette superfamily are present in a from paprika in the same laboratory and was demonstrated majority of human tumors and are an important final cause of to prevent the fragility of capillaries (4, 5). A large number therapeutic failure. Therefore, compounds which inhibit the of biologically active compounds (among them carotenoids) function of the MDR-efflux proteins may improve the cytotoxic were later extracted from vegetables, fractionated, action of anticancer chemotherapy. The effects of carotenoids characterized and systematically studied. were studied on the activity of the MDR-1 gene-encoded efflux The differentiation-inducing activity of carotenoids and pump system. The carotenoids, isolated from paprika and particularly vitamin A was described in the 30’s. Experimental other vegetables, were tested on the rhodamine 123 results suggested that various carotenoids serve as dietary accumulation of human MDR-1 gene-transfected L1210 chemopreventive agents (6), through their ability to quench mouse lymphoma cells and human breast cancer cells MDA- excited molecules and radicals. Such compounds can exert an MB-231 (HTB-26). Capsanthin and capsorubin enhanced the antitumor effect through different mechanisms, such as direct rhodamine 123 accumulation 30-fold relative to nontreated antiproliferative action, the suppression of oncogene lymphoma cells. Lycopene, lutein, antheraxanthin and expression, the inhibition of angiogenesis and the induction of violaxanthin had moderate effects, while alfa- and beta- differentiation, but a therapeutic effect was observed only in carotene had no effect on the reversal of MDR in the tumor precancerous conditions and promyelocytic leukemia (7). A cells. Apoptosis was induced in human MDR1 transfected preventive effect on age-related diseases was ascribed to mouse lymphoma cells and human breast cancer MDA-MB- carotenoids such as beta-carotene in vegetable food (8). 231 (HTB-26) cell lines in the presence of lycopene, zeaxanthin Indeed, a higher intake of carotenoid-rich foods is associated and capsanthin. The data suggest the potential of carotenoids with a reduced risk of the development of chronic diseases as possible resistance modifiers in cancer chemotherapy. such as cancer (9). A carrot-rich diet reduced tumor incidence in rats and mice treated with carcinogenic chemicals. In these studies, both beta- and alfa-carotene exhibited significant Paprika was imported to Europe from China and India and, antitumor-promoting activity in the two-stage carcinogenesis after the discovery of America, also from Mexico. An model (10). The complexes of carotenoids may restore important discovery in paprika research was made in the electrical conductivity (11) in various subcellular 1930s, when Albert Szent-Györgyi discovered the anti- compartments. Paprika extracts containing carotenoids were scorbutic substance ascorbic acid or vitamin C (1-3). Soon recently fractionated by Motohashi and his coworkers and some of the fractions proved to reverse multidrug resistance (MDR) of tumor cells (12). On the basis of the results mentioned above, and the Correspondence to: Joseph Molnár, Institute of Medical Microbiology contradictory results observed for saffron compounds (6) on and Immunology, University of Szeged, H-6720 Szeged, Dóm tér 10, Hungary. Tel: +36-62-545114, +36-62-545115, Fax: +36-62-545113, the ABC transporter responsible for the MDR of cancer e-mail: [email protected] cells, we studied the MDR-reversal effects of selected carotenoids isolated and identified from paprika and various Key Words: Multidrug resistance, apoptosis, carotenoids. plant species.

0258-851X/2004 $2.00+.40 237 in vivo 18: 237-244 (2004)

238 Molnár et al: Modulation of MDR and Apoptosis of Cancer Cells by Carotenoids

Figure 1. Carotenoids.

239 in vivo 18: 237-244 (2004)

Table I. The effects of carotenoids in reversing multidrug resistance. Table II. Induction of apoptosis in mouse lymphoma cells (transfected by human MDR-1 gene) with different carotenoids. Samples Conc. FSC SSC FL-1 Fluorescence (Ìg/mL) activity ratio Samples Conc. Early Total Cell death apoptosis apoptosis (%) Parental control 586 223 935 (Ìg/mL) (%) (%) Multidrug- 621 215 17 resistant control DMSO control 28. 9 9.3 1.99 Verapamil 5 622 217 99 5.73 Lycopene 20.0 41.8 55.2 0.05 Lycopene 2 618 205 19 1.10 ‚-Cryptoxanthin 20.0 52.9 8.6 0.12 20 622 200 168 9.70 ·-Cryptoxanthin 20.0 38.6 6.6 0.62 ‚-Carotene 2 597 215 11 0.66 Zeaxanthin 20.0 41.9 48.3 0.43 20 612 191 9 0.56 Lutein 20.0 57.2 20.1 0.35 ·-Carotene 2 575 213 13 0.76 Antheraxanthin 20.0 43.2 8.4 0.44 20 612 187 13 0.81 Violaxanthin 20.0 34.9 7.1 1.47 ‚-Cryptoxanthin 2 607 195 76 4.41 20 598 212 167 9.65 Capsanthin 20.0 40.8 19.7 0.51 ·-Cryptoxanthin 2 589 187 16 0.93 Capsorubin 20.0 31.3 40.9 0.11 20 571 289 76 4.42 M-627 50.0 7.7 87.6 1.40 Zeaxanthin 2 594 180 90 5.21 20 529 307 167 9.67 Lutein 2 597 183 149 8.61 20 529 242 174 10.06 Antheraxanthin 2 591 163 193 11.13 20 556 233 176 10.17 Reversal of MDR of tumor cells. The L5178 mouse T-cell lymphoma cell line was infected with pHa MDR1/A retrovirus as Violaxanthin 2 582 165 15 0.90 previously described (16). MDR-1-expressing cell lines were 20 574 206 103 5.97 selected by culturing the infected cells with 60 ng/mL colchicine. Capsanthin 2 538 255 727 30.98 The MDR and PAR (parent) cell lines were grown in McCoy's 20 476 340 647 27.59 5A medium with 10% heat-inactivated horse serum, L-glutamine Capsorubin 2 494 329 793 33.78 and antibiotics. The cells were adjusted to a density of 2 x 106/mL, 20 441 366 574 24.46 resuspended in serum-free McCoy's 5A medium and distributed Lycophyll 2 514 177 26 1.4 in 0.5 mL aliquots into Eppendorf centrifuge tubes. Various 20 449 123 471 25.2 volumes (2-20 ÌL) of the 1.0 mg/mL stock solutions of the tested DMSO control 20 485 625 25 1.10 compounds were then added and the cells were incubated for 10 min at room temperature. Ten ÌL (5.2 ÌM final concentration) rhodamine 123 indicator was then added and the cells were incubated for a further 20 min at 37ÆC, washed twice and resuspended in 0.5 mL phosphate-buffered saline (PBS) for analysis. The fluorescence of the cell population was measured by Materials and Methods flow cytometry, using a Beckton-Dickinson FACScan instrument. Verapamil was used as a positive control. The percentages of the Chemicals. Lycopene (Lycopersicon esculentum, tomato), capsanthin, control mean fluorescence intensity were calculated for MDR and capsorubin (Capsicum annuum, red paprika), antheraxanthin, parental cells as compared with untreated cells (16). The activity violaxanthin (yellow flowers of Viola tricolor), alfa-carotene, beta- ratio was calculated from the measured fluorescence values using carotene (Daucus carotta, carrot), alfa-cryptoxanthin, beta- the following formula: cryptoxanthin (yellow paprika, Valencia orange peels), zeaxanthin (Lycium halimifolium), lutein (Caltha palustris, Marsh Marigold) and MDR treated/MDR control lycophyll (Solanum dulcamara) had been isolated and identified R = earlier by our group (13-15). Stock solutions were prepared in parental treated/parental control DMSO. Assay of the effects of resistance modifiers on apoptosis induction: Cell cultures. L5178Y (parent) mouse T-cell lymphoma cells and Apoptosis induction in the presence of various carotenoids in the transformed subline with MDR (MDR1/A) were used. The human MDR1-carrying mouse lymphoma and human breast cancer cells were grown in McCoy’s 5A medium supplemented with 10% MDA MB 231 (HTB-26) cell lines was determined. The resistance heat-inactivated horse serum (16). The breast cancer MDA MB modifiers were applied before or after apoptosis induction with 231 (HTB-26) cell line was also used. These cells were grown in 12H-benzo(a)phenothiazine (M627). The cells were adjusted to a Leibovitz’s L-15 medium in the presence of 10% fetal bovine density of 2 x 105/mL and were distributed in 1.0 mL aliquots into serum. All media were supplemented with 2 mM L-glutamine and microcentrifuge tubes. M627 was applied at a final concentration antibiotics. of 50 Ìg/mL. The cells were incubated at 37ÆC for 45 min in the

240 Molnár et al: Modulation of MDR and Apoptosis of Cancer Cells by Carotenoids

Table III. The multidrug resistance-reversal effects of carotenoids on Table IV. Apoptosis induction by carotenoids on MDA-MB-231 (HTB26) MDA-MB231 (HTB-26) human breast cancer cells. human breast cancer cells.

Samples Conc. FSC SSC FL-1 Fluorescence Samples Conc. Early Total Cell death (Ìg/mL) activity (Ìg/mL) apoptosis apoptosis (%) ratioa (%) (%)

HTB-26 CONT 558 202 9.21 Cell control 9.07 15.64 4.79 Double staining HTB-26 +R123 557 197 1406 Lycopene 2.00 12.7 27.9 13.24 Verapamil 10 554 209 1376 0.978 ‚-Cryptoxanthin 2.00 5.9 11.5 1.06 Lycopene 4 560 218 1245 0.885 ·-Cryptoxanthin 2.00 10.5 8.0 2.01 ‚-Cryptoxanthin 4 544 212 1197 0.851 Zeaxanthin 2.00 9.1 35.3 6.18 ·-Cryptoxanthin 4 551 212 1349 0.959 Lutein 2.00 10.6 15.0 3.88 Antheraxanthin 2.00 17.3 10.1 1.31 Zeaxanthin 4 560 225 1210 0.860 Violaxanthin 2.00 7.7 22.7 10.10 Lutein 4 562 222 1318 0.936 Capsanthin 2.00 7.1 40.3 8.02 Antheraxanthin 4 563 214 1338 0.951 Capsorubin 2.00 8.2 17.3 7.04 Violaxanthin 4 548 200 1330 0.945 M-627 50.00 1.4 97.0 1.20 Capsanthin 4 557 215 1252 0.890 Capsorubin 4 547 213 1202 0.855 DMSO control 4 548 208 1306 0.928 aFluorescence activity ratio: mean fluorescence ratio for treated/untreated samples. Results

The drug uptakes by human MDR-1 gene-transfected mouse lymphoma cells were examined in the presence of selected presence of the inducer compound, the samples were then carotenoids. The carotenoids were classified into three different centrifuged and washed with serum-free medium, and the cells groups according to their MDR-reversal activity: inactive, were resuspended in 1.0 mL culture medium. The resistance moderately active or very active. As shown in Table I, alfa- and modifier drugs were added to the samples at a final concentration of 0.5 Ìg/mL and the cells were distributed in 24-well culture beta-carotene exhibited no effect. The most effective plates. After incubation for 24 h at 37 ÆC, the apoptosis of the cells compounds were capsanthin, lycophyll and capsorubin. was evaluated. Moderately effective carotenoids were violaxanthin, lycopene, When the resistance modifiers were applied before apoptosis lutein, zeaxanthin and antheraxanthin. The cell size decreased induction, the compounds were used at a final concentration of 1.0 by less than 5% following treatment with beta-cryptoxanthin, Ìg/mL. The cells were incubated in the presence of drugs for 45 lutein and lycophyll. Shrinkage was observed in the presence of min at 37ÆC, and then washed with serum-free medium and zeaxanthin. The results showed that the MDR-reversing resuspended in 1.0 mL culture medium. The apoptosis inducer M627 was added to the samples at a final concentration of 50 effect depended on the chemical structure. The early Ìg/mL. After incubation for 45 min at 37ÆC, the samples were apoptosis-inducing effects of carotenoids were high in the washed, resuspended in 1.0 mL culture medium and distributed in presence of lycopene, zeaxanthin and capsorubin, while 24-well culture plates. The cultures were further incubated for 24 h lutein and capsanthin had moderate activities. Alpha- and at 37ÆC and apoptosis was evaluated by flow cytometry. beta-cryptoxanthin, lutein, antheraxanthin and violaxanthin did not modify the total apoptosis, since the frequency was Apoptosis assay. The assay was carried out according to the the same as in the control untreated cells. (Table II). None Protocol of Alexis Biochemicals with minor modifications. The cells were transferred into small centrifuge tubes, centrifuged of the carotenoids tested induced noteworthy cell death in and resuspended in 1.0 mL binding buffer. After centrifugation, the cultures of MDR mouse lymphoma cells. 750 ÌL supernatant fluid was removed and 3 mL Annexin V- Besides mouse lymphoma cells, the rhodamine FITC was added to the samples. The samples were incubated at accumulation was also studied in human breast cancer HTB- room temperature for 30 min in the dark. Then 750 Ìl binding 26 cells in the presence of carotenoids. The rhodamine buffer was added to the samples and, after centrifugation, the accumulation was not modified in these cells after carotenoid cells were resuspended in 100 ÌL binding buffer and 1.0 ÌL from treatment (Table III) because these cells do not contain a a 1.0 mg/mL propidium iodide stock solution was added to the samples. The fluorescent activity (FL-1 and FL-2) of the cells MDR efflux pump. was measured and analyzed on a Becton-Dickinson FACScan When apoptosis and cell death were investigated in the instrument. human breast cancer cells, the highest rate of early

241 in vivo 18: 237-244 (2004) apoptosis induction was observed in the presence of inhibits cell growth by interfering with growth factor signal lycopene and antheraxanthin. The total apoptosis was transduction IGF in breast cancer, MCF7 and endometrial highest in the presence of lycopene, zeaxanthin and cancer (23, 24) due to a decrease in the IGF-induced capsanthin, comparable to that for the positive control, tyrosine phosphorylation of the insulin receptor substrate. 12H-benzo(a)phenothiazine (M627). In these cells, the It is assumed that the anticarcinogenic activity of some death rate was increased in the samples treated with natural carotenoids, such as beta-cryptoxanthine, is related lycopene, violaxanthin and capsanthin (Table IV). to the stimulation of the expression of antioncogenes (25). It is concluded that some carotenoids exert an MDR- A retinoic acid derivative was found to exert a high reversing effect and induce apoptosis is human MDR-1 antiproliferative action with similar inhibitory potency in gene-transfected mouse lymphoma cells. The same drug-resistant and parental cell lines of colon and mammary carotenoids were not able to modify the drug accumulation cancer (26). in drug-sensitive human breast carcinoma cells, while Various fractions of red and green paprika extracts apoptosis was induced in the breast cancer cells. It is reverse the MDR of cancer cells (12, 13). On the basis of interesting that both the MDR-reversing and the apoptosis- the effectiveness of hexane and acetone fractions of paprika inducing effects of the carotenoids tested in the experiments on the ABC transporter responsible for MDR-reversal, it on the two different cell lines were related to certain appeared worthwhile to study the effects of various well- chemical structures. The majority of carotenoids having defined carotenoids on the drug accumulation in tumor hydroxylation on the right six-membered ring exert a cells. The results show that the MDR-reversing effect moderate effect on the MDR reversal. In addition to the depended on the chemical structure. The explanation can hydroxylation on the left ring in the case of capsanthin and be approached from the aspects of submolecular biology capsarubin, a hydroxylation on the right five-membered ring and supermolecular chemistry. An aliphatic carotenoid is possibly responsible for the very high resistance reversal containing conjugated unsaturated rings (in a conjugated effect on the human MDR1 gene transfected mouse system) has a marked tendency for possible charged lymphoma cells (see chemical structures). transfer complex formation by acting as an electron acceptor which can modify the conformation and function Discussion of P-glycoprotein (27). Carotenoids were presumed to form charge-transfer Some carotenoids exhibit chemopreventive, anticarcinogenic, complexes with pgp170 responsible for the MDR in the resistance-modifying and apoptosis-inducing effects, tested mouse lymphoma cells. Spectral data showed that the depending on their chemical structures (12, 13, 17). carotenoids formed charge-transfer complexes with iodine, Epidemiological studies have indicated that a high due to a special-charge transfer transition between the consumption of vegetables and fruits rich in flavonoids and charge-transfer ground state and the excited state (28). carotenoids reduces the risk of various cancers. The common Moreover, carotenoid-protein complexes have low fruits and vegetables contain approximately 40-50 carotenoids activation energies (29). A chemical structure- dependent and many of them can be selected as chemopreventive agents resistance-modifying effect was found in our experiments on (17, 18). Apart from beta- and alfa-carotene, the rarer types human MDR-1 gene-transfected mouse lymphoma cells and of carotenoids from red paprika, e.g. prenigroxanthin (14), also when the rhodamine 123 uptake was increased in cycloviolaxanthin (13) and many others (15), have been myeloblasts from patients treated with all-trans-retinoic acid chemically characterized, but their effects on cancer cells have (30). Apart from the inhibition of MDR P-glycoprotein, the still not been studied in detail. Carotenoids such as lycopene non-MDR phenotype drug resistance of cancer cells was from tomato have been studied in vivo in cervical, breast, also reduced in vinca and taxol cross-resistant cells due to ovarian and colorectal cancer, and the results showed that the modification of the cytoskeleton reassembly mediated elevated serum carotenoid concentrations were associated by protein kinase-C (31). with a decreased risk of recurrence (19). There are data Some of the tested carotenoids modified the frequency of indicating that an increased consumption of carotenoids apoptosis in our experiments. Similar results were found reduces the risk of cancer, especially upper respiratory and earlier: all-trans-retinoic acid treatment of acute digestive tract cancers (20). promyelocytic leukemia cells induced apoptosis and In an investigation into the antiproliferative effect of suppression of Bcl-2 (30). The therapeutic effect of 13-cis- carotenoids, lycopene was found to be the most effective on retinoic acid appears to act through apoptotic processes in various prostate cancer cells (21). The growth inhibition was large cell lymphoma (32). A carotenoid-containing diet accompanied by a slowing-down of the cell cycle progression could suppress prostate cancer by inducing apoptotic cell through phases G1-S in mammary cancer MCF7 via the death with the involvement of Bcl-2 and Bax (33). Some reduction of cyclin D1 (22). It was found that lycopene structure-activity relationships concerning the importance

242 Molnár et al: Modulation of MDR and Apoptosis of Cancer Cells by Carotenoids of different forms of stereochemisty at the C9 double bond 13 Deli J, Molnár P, Tóth G, Baumeler A and Eugster CH: of carotenoids in conferring apoptotic activity have also Cycloviolaxanthin, ein neues Carotinoid aus Paprika (Capsicum been described (34). annum). Helv Chim Acta 74: 819-824, 1991. The different sensitivities of the cells to the various 14 Deli J, Molnár P, Matus Z, Traber B and Pfander H: "Prenigroxanthin" (beta-carotene-3,3,6’-triol) a novel carotenoid carotenoids can be explained by the differences in the from red paprika (Capsicum annum). Tetrahedron Letters 42: chemical structures of the carotenoids and their membrane- 1395-1397, 2001. crossing properties or the electronic distribution or the 15 Deli J, Molnár P, Osz E and Tóth G: Capsoneoxanthin, a new semiconductor properties of the compounds. 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27 Gutmann F, Jonhson C, Keyzer H and Molnar J: Charge 32 Su IJ, Cheng AL, Tsai FF and Lay JD: Retinoic acid-induced Transfer Complexes in Biological Systems, Marcel Dekker, Inc, apoptosis and regression of a refractory Epstein-Barr virus- New York, Basel, Honkong, 1997 p. 323 containing T cell lymphoma expressing multidrug-resistance 28 Huggins CM and Le Blanc OH: Electrical and magnetic phenotypes. Br J Haematol 85: 826-8 1993. properties of ‚-carotene triiodide. Nature 186: 552-553, 1960. 33 Kim HS, Christov K, Chen L, Ghosh L, Duncan C, Sharifi R 29 Eley DD and Snart RS: Conduction in chloroplast components. and Bowen P: Modulation of apoptotic cell death in prostate Biochim Biophys Acta 102: 379-385, 1965. tumors by tomato sauce supplementation. Abstract from 13th 30 Utsun C, Beksac M, Dalva K, Koc H, Konuk N, Ilhan O, Int Carotenoid Symposium, January 6-11, 2002, Honolulu. Ozcan M, Topcuoglu P, Sertkaya D and Hayran M: In vivo use Hawaii. USA. of all-trans-retinoic acid prior to induction chemotherapy 34 Simoni D and Tolomeo M: Retinoids, apoptosis and cancer. improves complete remission rate and increases rhodamine 123 Curr Pharm Des 17: 1823-37, 2001. uptake in patients with de novo acute myeloid leukemia. Med Oncol 19: 59-67, 2002. 31 Debal V, Breillout F and Manfait M: Concomitant decrease of resistance and modifications of the cytoskeleton after all- trans-retinoic acid and phorbol ester treatments in a Received September 8, 2003 navelbine-resistant bladder carcinoma cell line. Anticancer Revised December 15, 2003 Res 17: 1147-54, 1997. Accepted February 2, 2004

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