ANTICANCER RESEARCH 24: 865-872 (2004)

New Silicon Compounds as Resistance Modifiers against Multidrug-resistant Cancer Cells

J. MOLNAR1, I. MUCSI1, J. NACSA1, A. HEVÉR1, N. GYÉMÁNT1, K. UGOCSAI1, P. HEGYES2, ST. KIESSIG6, D. GAAL5, H. LAGE4 and A. VARGA3

1Albert Szent-Györgyi Medical Center, Faculty of Medicine, Institute of Microbiology and 2Faculty of Natural Science, Department of Organic Chemistry, University of Szeged, Hungary; 3Institute of Biology, Department of Molecular Parasitology and 4Medical Faculty, Institute of Pathology, University of Humboldt, Berlin,Germany; 5National Oncological Institute, Budapest, Department of Animal Trials, Hungary; 6Baxter (Immuno) AG, Heidelberg, Germany

Abstract. The efficiency of chemotherapy is often decreased lymphoma cells was increased without down-regulation of the by the development of resistance of cancer cells to cytostatic MDR1 gene expression tested by RT-PCR assay. The drugs. This phenomenon is in most cases caused by the activity rhodamine uptake was increased in L5178/MDR1 and Colo- of the various ABC transporters, multidrug-resistance (MDR) 320/MDR1-LRP, but not drug-sensitive human breast cancer gene-encoded p-glycoproteins, that pump anticancer drugs out MCF-7 and T47D, and L5178 mouse lymphoma parent cells of the cells. The inhibition of the activities of the MDR proteins in the presence of alis-409 and alis-421. The MRP-mediated MDR1 and MRP was investigated via the administration of carboxyfluorescein accumulation in HTB-26/MRP human two new organosilicon compounds, alis-409 and alis-421. The breast cancer cells and daunorubicin accumulation in human study was focused on the inhibition of MDR by blocking the stomach cancer cells 257P/MDR were not modified by these MDR1 gene expression and through the inhibition of the alis compounds. A synergistic interaction between epirubicin pump-function of mdr-p-glycoprotein, in human breast cancer and the silicon-substituted resistance modifiers was found only cell lines expressing mrp and prostate cancer cell line (PC-3). in MDR1-mediated MDR in the case of colo-320/MDR1-LRP Apoptosis induction and the interaction between epirubicin cells and mouse lymphoma cells transfected with the human and the silicon-substituted compounds were studied in human MDR1 gene. The results indicate that the organosilyl MDR-1gene-transfected mouse lymphoma and its parent cell derivatives specifically act on MDR1 p-glycoprotein 170. The line, Colo320/MDR-LRP and sensitive subline Colo205, by alis compounds act on pgp170 in a way which is similar to means of rhodamine 123 accumulation. The activity of MRP1 verapamil isomers. p-glycoprotein was studied in human breast cancer cell lines such as HTB-26/MRP1 and two MRP-negative breast cancer The proportion of chemotherapy-resistant cases in patient cell lines, T47D and MCF7, by carboxyfluorescein populations is continuously growing. The resistance of accumulation, and on a stomach cancer cell line. The activity microbial and cancer cells to chemotherapeutics is a great of MRP in 257P/MDR and its drug-sensitive derivative were challenge in current chemotherapy. One of the most studied in human stomach cancer cells by daunorubicin important forms of resistance is related to an increased drug accumulation in a flow cytometer. The two representative efflux, mediated by an ABC transporter, a p-glycoprotein, organosilicon derivatives, alis-409 and alis-421, showed called the mdr-1 efflux pump, which reduces the antiproliferative effects without apoptosis induction. The drug effectiveness of anticancer drugs against cancer cells. Other accumulation in the human MDR1 gene-transfected mouse p-glycoprotein-mediated drug transporters such as MRP, LRP and BCRP are also related to the increased drug efflux of cancer cells. New treatment procedures frequently produce treatment-resistant cells. The activity of the mdr- Correspondence to: Prof. Joseph Molnar, Institute of Medical membrane transporter protein can be separately inhibited Microbiology, Albert Szent-Györgyi Medical Center, University of Szeged, H-6720 Szeged, Hungary. Tel/Fax: 36-62-545114 / 36-62- in eukaryotic cells by resistance modifier compounds 545113, e-mail: [email protected] independently of the effect of gene regulation. The antiproliferative activities of substituted phenothiazines Key Words: Organosilicon, multidrug resistance, reversal. on various cell lines (such as breast and colon cancer,

0250-7005/2004 $2.00+.40 865 ANTICANCER RESEARCH 24: 865-872 (2004) leukemia, melanoma, ovarian and prostate cancer cells) were EPG85-257RNOV, and the classical MDR variant EPG85- recently observed. Colon cancer exhibits the highest 257RDB have been described previously (5,6). These tumor cells sensitivity against such compounds, followed by leukemia, were grown in Leibovitz L 15 medium (Bio Whittaker, Walkersville, Maryland, USA) supplemented with 10% fetal calf melanoma, prostate, breast and ovarian cancer cells. These serum (GIBCOBRL, Grand Island, NY, USA), 1 mM L-glutamine, data suggest some type-specific antitumor action of certain 6.25 mg/1 fetuin, 80 IU/l insulin, 2.5 mg/1 transferrin, 1 g/1 glucose, compounds. 1.1 g/1 NaHCO3, minimal essential vitamins and 20000 kIU/1 To achieve the most specific mdr-reversal effects, the trasylol in a humidified atmosphere of 5% CO2 at 37ÆC. role of the stereoselectivity of various chemosensitizers was Additionally, the classical MDR line EPG85-257RDB was grown earlier systematically studied (1,2). The pharmacologically by adding 2.5 p g/ml daunorubicin (Farmitalia Carlo Erba, inactive stereoisomers of CNS drugs can be regarded as Freiburg, Germany), and the atypical MDR variant EPG85- 257RNOV was grown at a mitoxantrone (Lederle, Wolfratshausen, possible resistance modifiers for the combination treatment Germany) concentration of 0.2 pg/ml. of drug-resistant cancer without any risk of unexpected side-effects in vivo (2). However, high doses of these Assay for antiproliferative effect. The effects of increasing compounds were required for daunorubicin accumulation concentrations of the drugs alone and their combinations on cell in MDR white blood cells of leukemic patients in ex vivo growth were tested in 96-well flat-bottomed microtiter plates. The studies(3). Based on previous results we decided to compounds were diluted in a volume of 50 ÌL 4 investigate the mdr reversal of the newly synthesized Then, 1 x 10 cells in 0.1 mL medium were added to each well, with the exception of the medium control wells. The culture plates compounds alis-409 and alis-421. In the present paper we were further incubated at 37ÆC for 72 h and at the end of the report on the anticancer and MDR-reversing effects of incubation period 20 ÌL MTT solution (from a 5 mg/mL stock) was these patented organosilicon compounds on mdr mouse added to each well. After incubation at 37ÆC for 4 h, 100 ÌL SDS lymphoma cells transfected with the human MDR1 gene, solution (10%) was measured into each well and the plates were colon cancer cells expressing MDR1-LRP and breast further incubated at 37ÆC overnight. The inhibition of the cell cancer cell lines. growth was determined by measuring the optical density at 550 nm (ref. 630 nm) with a Dynatech MRX vertical beam ELISA reader. Materials and Methods The percentage inhibition of cell growth was determined according to the formula: Chemicals. Compounds alis-409 (1,3-dimethyl-1,3-p-fluorophenyl- 1,3(3-morfolinopropyl)-1,3 disiloxan dihydrochlorid) and alis-421 OD sample - OD medium control 100 - ––––––––––––––––––––––––––––––––––––– x 100 (1,3-dimethyl-1,3-(4-fluorophenyl)-1,3-[3(4-buthyl)-(1piperazinyl)- [ OD cell control – OD medium control ] propyl]-1,3-disiloxan-tetrahydrochlorid). The compounds were originally synthesized by Hegyes et al. (4). The silicion substituted A checkerboard micro-plate method was applied to study the alis compounds and 12H-benzo(a)phenothiazine were dissolved in effects of drug interactions between resistance modifiers and DMSO by preparing stock solutions at a concentration 1.0 mg/ml. doxorubicin on cancer cells. Verapamil (Sigma, St. Louis MO, USA) was dissolved in water The effects of an anticancer drug such as doxorubicin and the (5.0 mg/ml). Annexin-V (human recombinant-FITC) (Alexis resistance modifiers alis-409 and alis-421 in combination were Biochemicals, ALEXIS DEUTCHLAND Gmbh, Grünberg, studied on various cancer cell lines. The dilutions of anticancer Germany) and epirubicin (Pharmacia and Upjohn, Milan, Italy) drug A were made in horizontal direction and the dilutions of were dissolved in deionized water. 3- [4,5-Dimethylthiazol-2-yl] resistance modifiers B vertically in the microtiter plate in 100Ìl 1-2,5-diphenyltetrazolium bromide (MTT) and sodium volume. The cell suspension in tissue culture medium was dodecylsulfate (SDS) were purchased from Sigma. The silyl- distributed to each well in 100 ÌL containing 5x104 cells. The plates compounds are patented in Germany: Patent no. 199 23 801. were incubated for 48 h at 37ÆC in a CO2 incubator. The cell growth rate was determined after MTT staining and the intensity Cell cultures. L5178 (parent) mouse T-cell lymphoma cells and the of the blue color was measured on a micro ELISA reader. Drug human MDR-1-transfected subline were cultured in McCoy's 5A interaction was evaluated according to the following system: medium supplemented with 10% heat-inactivated horse serum L- glutamine and antibiotics. Human cancer cell lines MCF-7, T47-D, FIC = ID / ID Colo 205 parent, Colo 320/MDR-LRP and PC-3 prostate cancer A 50A in combination 50A alone FIC = ID / ID cells were cultured in RPMI supplemented with 10% heat- B 50B in combination 50B alone inactivated fetal bovine serum, and HTB-26 breast cancer cells where FIC=fractional inhibitory concentration were cultured in Leibovitz L-15 medium supplemented with 10% heat-inactivated fetal bovine serum with 2 mM L-glutamine. Most FIX = FICA+FICB of these cell lines were maintained in a humified atmosphere of 5% FIX = 0.51-1 ADDITIVE EFFECT CO2 (except HTB-26, which do not need extra CO2) at 37ÆC. FIX < 0.5 SYNERGISM 257P/MDR and its parent 257 RD human stomach cancer cells 1 < FIX < 2 INDIFFERENT EFFECT were used for daunorubicin accumulation studies. The FIX > 2 ANTAGONISM establishment and characterization of the human gastric carcinoma cell line EPG85-257P, the corresponding atypical MDR subline where FIX=fractional inhibitory index

866 Molnar et al: Silicon Resistance Modifiers

Assay of drug activity on induction of apoptosis. The cells were removal of the medium and the addition of chilled 10% adjusted to a density of 2x105/ml and were distributed in 1.0 ml trichloroacetic acid. After incubation for 1 h at 4ÆC, wells were aliquots into Eppendorf centrifuge tubes. The apoptosis inducer washed 5 times with tap water, and cell-associated protein was benzo(a)phenothiazine was added to samples as a positive control stained by adding 0.4% SRB in 1% acetic acid for 10 min at room (5.0 Ìl from a 10 mg/ml stock solution, the final concentration temperature. Absorbance was measured at 540 nm after drying and being 50 Ìg/ml.) In the case of control cultures, 5 ml DMSO was resolubilization in 20 mM Tris-HCl (pH 10). For the determination added). The cells were incubated at 37ÆC for 45 min. Samples were of IC50 values of antitumor agents, cells were incubated with centrifuged, then washed with serum-free McCoy's medium and increasing concentrations of drugs, the absorbance difference of the cells were resuspended in 1 Ìl culture medium. The drugs used control cells without drug being set to 100%. Graphs of cell for the treatment were added to a volume of 1 ml from a 0.5 Ìg/mL survival against dose of antineoplastic agent were plotted, and ID50 stock solution to the samples, the final concentration of each drug values were calculated from multiple (at least three) independent being 0.5 mg/ml. After incubation for 24 h at 37ÆC, the cells were experiments for each cell clone. transferred from a 24-well plate into Eppendorf centrifuge tubes, centrifuged and resuspended in 1.0 ml annexin-buffer. The samples Results were mixed and centrifuged and 750 Ìl of the supernatant were then removed from each tube. Annexin-V-FITC (3-3 Ìl/ml sample) was added to the 250 Ìl sample remaining in the tubes. Controls In these experiments, the antiproliferative effects were without ∞nnexin-V were also prepared. The samples and controls measured on mouse lymphoma cells and the subline were incubated at room temperature for 30 min in the dark. Before transfected with the human MDR-1 gene. The 50% the measurement of fluorescence activity, 1.0 Ìl propidium iodide inhibitory concentrations (ID values) on mdr cells were (from a 1.0 mg/ml stock solution) was added to the samples. The 50 1.8 Ìg/mL for alis-409 and 3.1 Ìg/mL for alis-421. fluorescence activity of the cells was evaluated by flow cytometry in FL-1 and FL-2 in a Beckton Dickinson flow cytometer. From knowledge of the range of antiproliferative property, the effect of the two representative alis Assay for reversal of MDR in tumor cells. The L5178 mouse T cell compounds was studied on the activity of multidrug lymphoma cell line was infected with pHa MDR1/A retrovirus as transporter mdr1 pgp 170, responsible for the reduced drug previously described (7). MDR1-expressing cell lines were selected accumulation of tumor cells. In these experiments, the by culturing the infected cells with 60 ng/mL colchicine to maintain rhodamine 123 accumulation of drug-sensitive and MDR the expression of the MDR phenotype. The L5178 MDR and L5178Y parent cell lines were grown in McCoy’s 5A medium cell cultures was measured in the presence of the containing 10% heat-inactivated horse serum, L-glutamine and compounds. The two silicon derivatives markedly inhibited antibiotics. The cells were adjusted to a density of 2x106/mL, the mdr-p-glycoprotein-mediated efflux mechanism of the resuspended in serum-free McCoy’s 5A medium and distributed in mouse lymphoma cells, measured by flow cytometry. The 0.5 mL aliquots into Eppendorf centrifuge tubes. The tested results are shown in Table I. compounds alis-409 and alis-421 were added at various In RT-PCR experiments, the MDR1 gene expression was concentrations in different volumes (2.2-20.0 ÌL) of the 1.0-10.0 studied in the presence of the alis compounds. In the mg/mL stock solution and the samples were incubated for 10 min at room temperature. Next 10 ÌL (5.2 ÌM final concentration) presence of 380-500 Ìg/mL alis-409 and 421, there was no indicator rhodamine 123 was added to the samples and the cells change in the gene expression. The silyl derivatives only were incubated for a further 20 min at 37ÆC, washed twice and modified the activity of p-glycoprotein P170, but the MDR1 resuspended in 0.5 mL phosphate-buffered saline (PBS) for gene was not down-regulated. The alis compounds increased analysis. The fluorescence of the cell population was measured with the drug accumulation in human MDR1 gene-transfected a Beckton Dickinson FACScan flow cytometer. mouse lymphoma cells, providing evidence of the Verapamil was used as a positive control in the rhodamine 123 interaction between the p-glycoprotein and mdr-reversing exclusion experiments (8). The percentage mean fluorescence intensity was calculated for the treated parental and MDR cell alis compounds in flow cytometry. lines as compared with that of untreated cells. An activity ratio R To test the role of various multidrug transporters, the was calculated via the following equation (8) on the basis of the rhodamine-123 or carboxyfluorescein uptake by various measured fluorescence values epirubicin-sensitive human breast and prostate cancer cell lines was studied systematically in the presence of the two mdr treated /mdr control R = –––––––––––––––––––––––––––––––––– silicon-substituted compounds; verapamil, or indomethacin parental treated / parental control in the MRP study, served as controls. The rhodamine-123 accumulation in the two different drug-sensitive cell lines Cytotoxicity assay. Chemoresistance was tested by using a decreased with increasing the concentrations of alis proliferation assay based on sulforhodamine B (SRB), a protein- compounds. Similarly, verapamil reduced the rhodamine binding reagent (9), as described previously (10, 11). Briefly, stomach cancer cells were distributed into 96-well plates at a uptake by the MCF7 and PC3 cells, most likely as a density of 200 cells/well. For adhesion, the cells in the logarithmic consequence of a general membrane effect on the cells. growth phase were incubated for 48 h prior to drug application. Our results showed that in these cells the mdr transporter After 5 days of drug treatment, the assay was terminated by is not functional (Table II). Since the rhodamine

867 ANTICANCER RESEARCH 24: 865-872 (2004)

Table I. Reversal of multidrug resistance in MDR-1-transfected mouse Table II. Drug accumulation of MCF-7 human breast cancer cells in the lymphoma and colon cancer cells in the presence of alis-compounds. presence of alis compounds.

Samples Concentration Flow cytometry FL-1 Fluorescence Samples Concentration Flow cytometry FL-1 Ìg/ml FSC SSC activity ratio Ìg/ml FSC SSC

Parent cells 613 1052.7 MCF-7 522 204 1015 Mdr cells 648 10.6 Verapamil Verapamil 5.0 637 251 37.8 3.6 1 mg/ml 5 543 223 672 control DMSO 20 533 224 672 Alis-409 0.02 646 290 27.6 2.6 Alis-409 2 541 224 723 Alis-409 0.2 636 281 149.9 14.1 PC-3 445 190 848 Alis-409 2.0 639 284 1430.2 134.5 Pipolphen 2 448 200 482 Alis-421 0.02 638 287 32.3 3.0 Melipramin 2 441 199 448 Alis-421 0.2 640 297 188.7 17.7 Melipramin 20 447 190 281 Alis-421 2.0 630 287 1399.5 131.7 Alis-409 2 442 204 514 Alis-409 20 399 168 276 HTB-26 cells 498.5 239.8 374.3 Indomethacin 10.00 589.0 224.7 724.0 3.25 control Alis-409 0.40 506.1 209.7 208.2 0.9 Alis-409 4.00 514.5 204.0 220.1 0.9 Table III. Effects of alis compounds on 12H-benzo(a)phenothiazine- DMSO control 501.8 202.6 200.1 1.0 induced apoptosis in mdr K-562 and mouse lymphoma cells. Indomethacin 10.00 542.1 315.8 815.3 1.5 control Samples Total Early Cell death Alis-421 4.00 543.3 317.7 455.8 0.8 apoptosis % apoptosis % % Alis-421 40.00 402.6 283.5 383.7 0.7 DMSO control 500.1 294.0 427.1 0.7 K 562 cell control 1.09 0.14 0.10 K 562 + 12H- 18.2 1.2 23.3 Colo-320 cells 536..1 219.7 46.5 benzo(a)phenothiazine (1.0 mg) Verapamil 5.00 559.8 218.7 570.9 12.2 K 562 + Alis-409 control (0.5 Ìg) 0.75 0.07 2.76 Alis-409 0.40 587.9 209.7 228.9 4.9 K 562 + Alis-421 Alis-409 4.00 587.8 219.8 564.0 12.1 (0.5 Ìg) 0.82 0.06 2.31 Alis-409 40.00 591.7 207.9 863.5 18.5 Mouse lymphoma cells control 0.63 0.09 0.42 DMSO control 560.8 225.5 33.1 0.71 Mouse lymphoma cells + 11.34 2.84 18.3 Verapamil 5.0 462.5 142.2 821.2 3.73 12H-benzo(a)phenothiazine control Mouse lymphoma cells + Alis-409 Alis-421 4.00 455.3 147.7 1029.0 4.67 (0.5 Ìg) 1.03 0.19 3.66 Alis-421 40.00 475.3 1285 1112.6 5.05 Mouse lymphoma cells+ Alis-421 DMSO control 470.5 148.3 202.7 0.92 (0.5 Ìg) 2.78 0.61 7.78

accumulation in these sensitive human cell lines decreased for alis-409 was 5.0 Ìg/ml, that for alis-421 was 5.0 Ìg/mL, following the treatment, a direct membrane effect or and for epirubicin was 3.12-6.25 Ìg/mL on mdr cells in apoptosis induction was assumed. However, the data vitro. The ID50 for epirubicin was decreased to 0.78-1.5 showed that the two tested compounds are not able to Ìg/mL in the presence of 1.25 Ìg/mL alis-409. Alis-421 induce apoptosis in the applied concentration range (0.1 to had a similar, but less marked effect relative to that of 2.0 Ìg/mL) (Table III). alis-409. Based on the synergism found between epirubicin Since the mdr-reversal effect was observed at a and the mdr-reversing compounds alis-409 and alis-421 in concentration 1.0 Ìg/ml (1.0 mg/kg body weight in vivo), vitro, the nature of the interaction between epirubicin and the antiproliferative effect of the known anticancer agent the resistance modifiers was also studied on non-MDR epirubicin was studied in the presence of different cells such as MCF-7, T-47D and HTB-26 breast cancer concentrations of alis-409 and alis-421. The results cell lines (Table IV). No significant interaction was found obtained in these experiments provided evidence of between the alis compounds and daunorubicin in these interaction between epirubicin and these resistance non-MDR human tumor cells. The two new organosilicon modifiers in vitro. In the antiproliferative assay, the ID50 compounds exerted strong inhibitory effects on the efflux

868 Molnar et al: Silicon Resistance Modifiers pump activity of the MDR (mdr-1) tumor cells such as Table IV. Antiproliferative effects of combinations of epirubicin and two mouse lymphoma and colo-320 cell lines (Table V). The resistance modifiers on various cell lines. organosilicon compounds enhanced the antiproliferative Cell Treatment ID values Comments effect of daunorubicin on the human MDR-1-transfected 50 mouse lymphoma T-cell line L/5178 and colo-320/MDR- L5718/MDR LRP cells, but no effects were found for the resistant epirubicin 3.12- 6.25 human gastric cancer cells (Table VI) or HTB-26 human Alis-409 5.0 Alis-421 5.0 breast cancer cells expressing in vitro (Table IV). The Alis-409+epirubicin 1.25 +0.78 compounds did not induce apoptosis on tumor cells. Alis-421+epirubicin 1.25 +1.50 synergism MCF-7 Discussion epirubicin 0.78-1.9 Alis-409 15.0-20.0 Some organosilicon compounds are well known cytostatic Alis-421 8.0 - 10.0 Alis-409 + epirubicin 9.0 + 1.9 drugs: 2.6-cis-diphenylcyclotetrasiloxane (Cisobitan) was Alis-421 + epirubicin 1.25+ 1.5 no interaction found to be partially effective in the in vivo treatment of patients with prostate carcinoma (12-14). Cisobitan, with the T-47D generic name quarosilan, is an organosilicon drug with high epirubicin 1.50 estrogenic activity. Other synthesized fluoro-organo-silicon Alis-409 10.0-15.0 Alis-421 8.0 - 10.0 complexes displayed cytotoxic effects in human ovarian Alis-409 + epirubicin 1.25 + 1.5 carcinoma cell cultures in vitro and one compound Alis-421 + epirubicin 1.25 + 1.5 no interaction prolonged the life of mice with MX-11 tumor (15). A similar silicon compound, as 2-piperidoethyl- HTB-26/MRP1 phenyldimethylsilane, exhibited an inhibitory effect on Ehrlich epirubicin 0.5- 1.6 Alis-409 8.0- 10.0 sarcoma-180 and Lewis lung carcinoma in mice and rats. This Alis-421 10.0- 25.0 latter compound was also able to increase the delayed type Alis-409 + epirubicin 2.50 + 0.5 cellular hypersensitivity (16). The high toxicity and side-effects Alis-421 + epirubicin 12.0 + 0.15 no interaction of organo-silicon compounds (17) limited their application in the therapy of cancer. Organo-silicon compounds were Colo320/MDR1-LRP epirubicin 0.25-0.6 isolated from the mycobacterium Sorangium cellulosum and Alis-409 25.0-50.0 their antitumor effect was shown (18). It turned out that these Alis-421 20.0-50.0 natural products (epothilones A and B) were cytotoxic Alis-409 + epirubicin 12.5 + 0.15-0.30 macrolides. A total synthesis of the compounds was published Alis-421 + epirubicin 25.0 + 0.15-0.30 synergism by Zhu and Panek (19). Epothilones have a taxol-like antitumor mechanism and they are also effective against taxol- resistant tumor cell lines (20). Taxanes and epothilones A and properties. A combination of the new silicon compounds in low B kill cancer through the induction of tubulin polymerization concentration with cytostatic drugs resulted in a reversal of the and microtubule stabilization. MDR of cancer cells in vitro and also enhanced the cytotoxic Anticancer chemotherapeutic agents lose their effect because effect. The antiproliferative effect of silicon-substituted mdr-gene expression is induced in tumor cells (21). The compounds may also contribute to the antitumor action of the possible application of organo-silicon compounds is a new combination chemotherapy. Following these in vitro studies, the approach for the reversal of MDR (21). 1,3-Dimethyl-1,3-bis(4- alis compounds can be recommended for animal experiments fluorophenyl)-1,3-bis(3-morpholinopropyl)-disiloxane-dihydro as promising MDR reversal agents. chloride (alis-409) and 1,3-dimethyl-1,3-bis(4-fluorophenyl)-1,3- bis{3-[1(4-butylpiperazinyl}]propyl)disiloxane-tetrahydrochlorid Acknowledgements (alis-421) display marked effectiveness for reversal of the MDR in mdr-1 cancer cells. The activity of the efflux pump was This study was supported by the Szeged Foundation of Cancer inhibited directly without any effect on the gene expression of Research (Szegedi Rákkutatatásért Alapitvány), Hungary and the mdr-1. The inhibitory effects of these compounds for the efflux COST B16 Action of the EU Commission. pump activity may facilitate a decrease of the effective dose of anticancer chemotherapeutics by reducing the adverse effects References of anticancer chemotherapy in their application in clinical 1 Szabo D and Molnar J: The role of stereoselectivity of practice. The chemical stability and good water-solubility of alis- chemosensitizers in reversal of mouse lymphoma cells. 409 and alis-421 constitute positive pharmacologically Anticancer Res 18: 3039-3045, 1998.

869 ANTICANCER RESEARCH 24: 865-872 (2004)

Table V. MDR- reversal effect of alis-409 and alis-421 on Colo320 and Colo205 cells.

Samples Dye Final FSC SSC FL-1 Fluorescence Peak concentration activity Ch 2003.02.21. ÌL 10 ÌL Ìg/ÌL ratio

1 Colo 205 - R 123 - 551.48 246.72 895.33 873 2 Colo 205 - - - 556.58 240.60 2.2 1 3 Colo 320 - R 123 - 536.12 219.76 46.58 29 4 Colo 320 - - - 555.21 224.70 2.50 1 5 Colo-320 5 R 12310 559.85 218.70 570.94 12.25 509 + verapamil 6 Colo-320 20 R 123 - 560.82 225.51 33.14 0.71 20 + DMSO 7 Cool 320 2 R 123 0,4 5 209.74 228.92 4.91 224 + Alis-409 87.94 8 Cool 320 20 R 123 4 219.88 564.04 12.10 572 + Alis-409 587.81

9 Colo-320 20 R 123 40 591.79 207.96 863.56 18.53 784 + Alis-409 10 Colo-320 2 R 123 4 455.3 141.8 1029 4.67 1009 + Alis-421 11 Colo-320+Alis-421 20 R 123 40 475.3 475.3 1112.6 5.05 1064 12 Colo-205 control 506.0 185.5 874.4 777 13

Table VI. Effects of alis- 409 and alis-421 on drug sensitivity of gastric 2 Varga A, Sabat R, Mucsi I, Flores VC, Kaiser HE and Molnar carcinoma cells. J: Effects of butaclamol, clopentixol, mepromazine and cannabinol stereoisomers on apoptosis induction. Anticancer Cell line EPG85-257P1 Res 21: 2709-2712, 2001. ID50 ID50 3 Szabo D, Szabo G, Ocsovszki I, Aszalos A, Molnar J: Antipsychotic drugs reverse multidrug resistance of tumor cell lines Daunorubicin and human AML cells ex-vivo. Cancer Letters 139: 115-119, 1999. Control 9.0 ng/ml 4 Patent: Hegyes P, Molnar J, Mucsi I, Hever A, Szabo D, Kiesig 0.1 ÌM Alis-409 9.25 ng/ml S, Lage H, Gaal D and Nacsa J: Substituted disiloxanes method 0.3 ÌM Alis-421 9.25 ng/ml for the production thereof and the use thereof for reversal of multidrug resistance (MDR) PCT/DE00/04110, 2000. EPG85-257RDB2 5 Dietel M, Arps H, Lage H and Niendorf A: Membrane vesicle Daunorubicin formation due to acquired mitoxanthrone resistance in human Control 6.8 Ìg/ml gastric carcinoma cell line EPG85-257. Cancer Res 50: 6100- Alis-409 6.4 Ìg/ml 6106 1990. Alis-421 5.8 Ìg/ml 6 Lage H and Dietel IYI: Effect of the breast cancer resistance protein on atypical multidrug resistance. Lancet Oncol 1: 169- EPG85-257P1 175, 2000. Mitoxantrone 7 Cornwell MM, Pastan I and Gottesmann MM: Certain Control 42.5 ng/ml Alis-409 37.0 ng/ml calcium channel blockers bind specifically to multidrug- Alis-421 50.0 ng/ml resistant human KB carcinoma membrane vesicles and inhibit drug binding to P-glycoprotein. J Biol Chem 262: 2166-2170, EPG85-257RNOV3 1987. Mitoxantrone 8 Gruber A, Peterson C and Reizenstein P: D-verapamil and L- Control 5.9 Ìg/ml verapamil are equally effective in increasing vincristine Alis-409 4.8 Ìg/ml accumulation in leukemic cells in vitro. Int J Cancer 41: 224- Alis-421 5.4 Ìg/ml 226, 1988. 9 Skehan P, Storeng R, Scuctiero D, Monks A, McMahon J, 1 parental, drug sensitive Yistica D, Warren IT, Bokesch H, Kenney S and Boyd MR: 2 "classical", P-gp-dependent MDR New colorimetric cytotoxicity assay for anticancer drug 3 "atypical", P-gp-independent MDR screening. J Natl Cancer Inst 82: 1107-1112, 1990.

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