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Anti-Cancer Agents in Medicinal Chemistry, 2012, 12, 000-000 1 Anticancer Effects of the Organosilicon Multidrug Resistance Modulator SILA 421 Ulrike Olszewski1,*, Robert Zeillinger1, Meltem Demirel Kars2, Attila Zalatnai3, Jozsef Molnar4 and Gerhard Hamilton1 1 Ludwig Boltzmann Society, Cluster of Translational Oncology, Vienna, Austria; 2Advanced Technology Research and Application Center, Selcuk University, Konya, Turkey; 31st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary; 4Department of Medical Microbiology, University of Szeged, Szeged, Hungary Abstract: 1,3-dimethyl-1,3-bis(4-fluorophenyl)-1,3-bis{3-[1(4-butylpiperazinyl)]-propyl}-disiloxan-tetrahydrochlorid (SILA 421) is a compound that was developed as modulator of the ABC cassette transporter P-glycoprotein. Furthermore, it exerted antimicrobial toxicity, vascular effects, downregulation of chaperone induction and plasmid curing in bacterial cells. Here, this drug was found to possess cytotoxic activity against a panel of human cancer cell lines that do not overexpress P-gp, with 50% inhibitory concentrations ranging between 1.75±0.38 M for GLC14 small cell lung cancer and 34.00±4.75 M for PC-3 prostate cancer cells. HL-60 leukemia and MDA-MB-435 breast cancer cells exhibited cell cycle arrest and apoptotic cell death in response to SILA 421. Assessment of global gene expression of SILA 421-treated HL-60 cells was employed to identify cellular pathways affected by the compound and revealed disturbance of DNA replication, transcription and production of apparently misfolded proteins. Endoplasmatic reticulum stress and downregulation of cell cycle, cellular repair mechanisms and growth factor-related signaling cascades eventually resulted in induction of apoptosis in this cell line. Reversal of resistance to taxanes, which had been reported for SILA 421 and the related molecule SILA 409, may be linked to downregulation of gene expression of kinesins, in addition to the well-established P-gp-inhibitory effect of SILA compounds. Interference with DNA replication and transcription seems to be the common denominator of antimicrobial activity and plasmid curing, as well as anticancer toxicity in human cell lines. Thus, in consideration of the full range of putative cellular targets found in the present work, the application of these SILA compounds for treatment of tumors should be further evaluated. Keywords: Annexin V, cell lines, cellular pathways, cytotoxicity, drug resistance, gene expression, HL-60, MDR modulator, organosilicon, P-glycoprotein, phenothiazine, Reactome, SILA 421. INTRODUCTION Multidrug resistance (MDR), a principal mechanism by which several cancers develop resistance to chemotherapeutics, is a major obstacle to the success of cytotoxic treatment of many tumor types. Since the first characterization of P-gp in 1979 the ATPbinding cassette (ABC) proteins have constituted targets of intense research, and many approaches to cancer therapy aim at the discovery of novel modulators capable of reversing MDR. The two patented organosilicon (SILA) compounds 1,3-dimethyl-1,3-bis (4fluorophenyl)-1,3-bis (3-morpholino-propyl) disiloxan-dihydrochlorid (SILA 409; patent designation: ALIS-409; German patent DE 1999 23801.4: PCT/DE 00/04110) and 1,3-dimethyl-1,3-bis(4fluorophenyl)-1,3-bis{3-[1(4-butylpiperazinyl)]-propyl}-disiloxantetrahydrochlorid (SILA 421; patent designation: ALIS-421; European Patent EP 1 432 717 B1; PCT/DE2000/004110) were developed as MDR modulators [1]. First evidence in a prostate, colon, stomach and three breast cancer cell lines indicated that SILA 409 and SILA 421 acted on P-gp specifically by direct inhibition, without having any effect on MDR1 gene expression [2]. Typical concentrations of the two compounds for reversal of MDR in mouse lymphoma cells transfected with the human MDR1 gene were 2.25 M for SILA 409 and 3.88 M for SILA 421, respectively. Both SILA drugs induced apoptosis at a concentration of 0.625 M to a minor extent in these cells. Additionally, they were shown to be capable of reversing MDR by inhibition of P-gp but not MRP1 in MCF-7 variants made resistant to paclitaxel, docetaxel, doxorubicin and vincristine by inhibition of P-gp but not MRP1 [3]. SILA 409 or SILA 421 in combination with either of the above-mentioned anticancer drugs resulted in synergistic or additive antiproliferative effects on the resistant MCF-7 sublines, respectively. SILA 409 was further tested in human pancreatic * Address correspondence to this author at the Cluster of Translational Oncology, Ludwig Boltzmann Society, Nussdorferstrasse 64/6, A-1090 Vienna, Austria; Tel: 43 1 40400 6627; Fax: 43 1 40400 6627; E-mail: [email protected], [email protected] 1871-5206/12 $58.00+.00 cancer xenografts (PZX-40/19G) and revealed delayed tumor growth, increased apoptosis, no effect on the mitotic index, reduced expression of P-gp and absence of toxicity on normal tissues [4]. Investigation of SILA 409 and SILA 421 demonstrated that K+induced contraction in rat aorta rings was significantly antagonized at higher drug concentrations and extracellular Ca2+ influx in single rat tail artery myocytes was blocked at lower doses [5]. Antimicrobial evaluation of SILA 421 showed greater efficacy against drug-resistant H37Rv strains of Mycobacterium tuberculosis in vitro than the related compound SILA 409 in vitro [4, 6] SILA 421 also conveyed killing potential to macrophages that had phagocytosed bacteria ex vivo, while being nontoxic against human lymphocytes [7]. Elimination of plasmids from highly tetracyclineresistant Escherichia coli strains was 105-fold increased. A histological assessment of 10 mg/kg body weight SILA 421 s. c every second day for 34 days revealed no apparent toxicity on vital organs in a mouse model [4-6]. Our study aimed at the assessment of the cytotoxicity of SILA 421 against a panel of normal and cancer cell lines, not selected for higher drug resistance, in order to search for possible effects apart from the modulation of MDR. Furthermore, analysis of SILA 421induced alterations of gene expression of HL-60 promyelocytic leukemia cells was performed to characterize the specific genes and cellular pathways affected by treatment with this compound to elucidate mechanisms of cytotoxicity and putative additional synergism with chemotherapeutics distinct from the modulation of MDR. MATERIALS AND METHODS Chemicals Unless noted otherwise, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The test compounds 1,3dimethyl-1,3-bis(4-fluorophenyl)-1,3-bis (3-morpholino-propyl) disiloxan dihydrochlorid (SILA 409; MW 621.7) and 1,3-dimethyl© 2012 Bentham Science Publishers 2 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 0 Olszewski-Hamilton et al. 1,3-bis(4-fluorophenyl)-1,3- bis{3-[1(4-butylpiperazinyl)]-propyl} disiloxan tetrahydrochlorid (SILA 421; MW 804.9) were synthesized by Hegyes according to the patent description and kindly provided by Prof. J. Molnar (Szeged, Hungary) [1]. Their molecular structures are presented in Fig. (1). Stock solutions of SILA 421 or doxorubicin were prepared in DMSO or 96% ethanol, respectively, and stored at -20°C. wavelengths of 488 and 675 nm, respectively. The proportion of apoptotic subG1 cells was obtained from the logarithmic PI histograms and percentages of cells in cell cycle phases G1/0 (resting), S (DNA synthesis) and G2M (mitotic) were calculated from the linear PI histograms using the MultiCycle AV software (Phoenix Flow Systems, San Diego, CA, USA). Experiments were done in duplicate. Cell Lines and Culture Conditions All cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA), except the doxorubicinresistant HL-60DX cells provided by the Department of Pathophysiology and Allergy Research (Medical University of Vienna, Austria) [8]. Cells were grown in RPMI-1640 bicarbonate medium (Seromed, Berlin, Germany) supplemented with 10% fetal bovine serum (Seromed), 4 mM glutamine and antibiotics (10x stock formulated to contain ~5,000 units penicillin, 5 mg streptomycin and 10 mg neomycin/ml), checked for mycoplasma contamination (Mycoplasma PCR ELISA, Roche Diagnostics, Vienna, Austria) and subcultured twice a week. Attached cells were subcultured by trypsinization. Annexin V-FITC Apoptosis Assay Cells were incubated with 25 M SILA 421 in six-well plates for 24 h and viable/apoptotic/necrotic cells were thereafter detected using the Annexin V-FITC Apoptosis Detection Kit APOAF (Sigma-Aldrich) in flow cytometry (Cytomics FC500, Beckman Coulter). The test was carried out according to the manufacturer´s instructions. Briefly, cells were washed twice with PBS and resuspended in binding buffer at a concentration of ~1x106 cells/ml. 5 l annexin V-FITC conjugate and 10 l PI solution were added to aliquots of 500 l cell suspension and incubated protected from light at room temperature for exactly 10 min. Then, fluorescence of the cells was immediately determined in flow cytometry. Green staining indicates cells in the apoptotic state, viable cells show no staining, necrotic cells appear red and finally, cells in the late apoptotic/necrotic state are stained red and green by both PI and annexin V-FITC conjugate, respectively. Chemosensitivity Assays 1x104 cells in 100 l medium per well were distributed in 96well microtiter plates (Greiner, Kremsmuenster, Austria) and substances to be tested added in another 100 l. SILA 421 was serially diluted in twofold steps in triplicate. The microtiter plates were incubated under tissue culture conditions (37°C, 5% CO2, 95% humidity) for four days and cell viability was measured using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (EZ4U, Biomedica, Vienna, Austria). This assay quantifies mitochondrial activity and therefore cell viability by the generation of a formazane dye from tetrazolium salt by mitochondrial reduction. Optical density was measured using a microplate reader at 450 nm with an empty well as reference. Values obtained from control wells containing cells and media alone were set to 100% proliferation. Cell Cycle Analysis 1x106 cells per well were incubated with SILA 421 in six-well plates for four days. Harvested cells were washed with PBS and fixed with 70% ethanol at -20°C for 30 min, washed again, transferred into staining solution (20 g/ml propidium iodide (PI), 5 g/ml ribonuclease A, 0.05% Nonidet P40 in PBS) and incubated at room temperature overnight. Washed cells were analyzed by acquisition of 1x104 cells in flow cytometry (Cytomics FC500, Beckman Coulter, Krefeld, Germany) at excitation and emission Genome-wide Gene Expression Analysis HL-60 cells, either untreated or incubated with 1.25 M SILA 421 in 175 cm2 flasks under tissue culture conditions for four days, were harvested and pellets of approximately 35x106 cells stored frozen at -80°C. Briefly, lysis with extraction buffer (4 M guanidine isothiocyanate, 0.5% sodium N-lauroylsarcosinate, 10 mM EDTA, 5 mM sodium citrate, 100 M -mercaptoethanol) was performed at 4°C, and DNA and RNA of the lysates were separated by cesium trifluoroacetate ultracentrifugation. RNA was washed with ice-cold 96% ethanol and dissolved in water. Measurements of the optical density at 260/280 nm proved content and purity of the RNA. Gene expression analysis was performed using the Applied Biosystems Human Genome Survey Microarray V2.0 (Applied Biosystems, Foster City, CA, USA). Therefore, 2-5 g mRNA (2050 g total RNA) were reverse transcribed to first-strand cDNA and labeled with digoxigenin-UTP according to the Applied Biosystems Chemiluminescent Reverse Transcription protocol. Hybridization of cDNA and microarray analysis was performed pursuant to the Applied Biosystems Chemiluminescence Detection Kit protocol and by use of the Applied Biosystems 1700 Chemiluminescent Microarray Analyzer. Data were processed by filtering and quantile normalization. Microarray probe identities were allocated to the respective gene designations using the microarray data information provided by Applied Biosystems. Pathway analysis was carried out by help of the Reactome database available at http://www. reactome.org/. Statistics Statistical analysis was performed using two-tailed Student’s ttest for normally distributed samples (*p<0.05 was regarded as statistically significant). Values are shown as mean±SD. RESULTS Fig. (1). Molecular structures of the two related organosilicon compounds SILA 409 and SILA 421. Cytotoxic Effect of SILA 421 in Wildtype or Doxorubicinresistant HL-60 Cells MTT proliferation tests were performed in order to investigate the cytotoxic activity of SILA 421 in HL-60 cells with normal doxorubicin-sensitivity (HL-60) as well as in resistant HL-60 cells (HL-60DX). Therefore, cells were incubated with nine twofold dilutions of 0.078-200 ng/ml doxorubicin, either in absence or in presence of 0.625 M SILA 421 (Fig. 2). Doxorubicin exerted Anticancer Effects of the Organosilicon Multidrug Resistance considerable dose-dependent antiproliferative activity in the wildtype cells which was not modulated in the presence of SILA 421 (Fig. 2, left). By contrast, an IC50 value was not obtained within the doxorubicin concentration range used in the resistant HL-60DX cell line, proving the high chemoresistance of these cells. Albeit the dose-response curve of HL-60DX cells was not shifted by SILA 421 at dilutions below 12.5 ng/ml doxorubicin, higher concentrations of doxorubicin (>12.5 ng/ml) applied simultaneously with 0.625 M SILA 421 decreased the proliferation of the resistant cells by approximately 5.7-14.5% (Fig. 2, right). These results corroborate the modulatory activity of SILA 421 in MDR-resistant cells. Screening of the Cytotoxic Effect of SILA 421 in a Panel of Cell Lines Cell proliferation in response to SILA 421 was assessed by MTT proliferation tests in a panel of 22 cell lines including Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 0 3 untransformed human embryonic kidney HEK-293 cells as well as leukemic (HL-60, U-937, K562), colon cancer (COLO 205, SW480, COLO 320 DM), pancreatic cancer (BxPC-3, MIA PaCa2), breast cancer (MDA-MB-435, T47D), prostate cancer (PC-3: hormone-insensitive; LNCaP: hormone-sensitive), medullary thyroid cancer (MTC-SK), glioblastoma (U373 MG), ovarian cancer (SKOV-3) and lung cancer (A549: non-small cell lung carcinoma (NSCLC); GLC14, GLC19, NCI-H526, DMS 153, NCIH417: small cell lung carcinoma (SCLC)) cells. They were incubated with twofold dilutions of 0.098-50 M SILA 421 for four days, and the resulting dose-response curves served for the calculation of the IC50 values shown in Fig. (3). Generally, the cancer cells revealed considerable sensitivity to SILA 421, with highest effects observed in SCLC (GLC14: IC50 1.75±0.38 M NCI-H417: IC50 10.63±0.75 M) and gastrointestinal (COLO 320 DM: IC50 4.38±0.53 M - SW480: IC50 10.38±0.88 M) cancer cells, while HEK-293 cells exhibited a higher IC50 value of Fig. (2). Dose-response curves for HL-60 wildtype (left) or HL-60DX doxorubicin-resistant cells (right) by simultaneous application of doxorubicin and 0.625 M SILA 421. Doxorubicin was serially diluted in nine twofold dilution steps. Values are presented as mean±SD (n=3). Differences for HL-60 wildtype cells and SILA 421 were not significant, in contrast to the HL-60DX cells revealing significant differences for SILA 421 at doxorubicin concentrations 25 ng/ml. Fig. (3). Chemosensitivity of a panel of one normal as well as cancer cell lines to SILA 421. The IC50 values were derived from dose-response curves of MTT proliferation assays. The compound was serially diluted in eight twofold steps. Values are presented as mean±SD (n=3). With exception of the K562, MDAMB-435, SKOV-3 and PC-3 cells, IC50 values of all other cell lines were significantly different from the normal HEK-293 cells. 4 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 0 19.88±1.63 M. Overall, the IC50 values of SILA 421 for the tumor cell lines ranged between 1.75±0.38 M for GLC14 and 34.00±4.75 M for PC-3 cells. The hormone-sensitive LNCaP prostate cancer cell line proved to be susceptible to SILA 421 (IC50 10.13±0.75 M) in contrast to the hormone-resistant PC-3 cells. SILA 421 moreover exhibited antiproliferative effects in both NSCLC and SCLC cell lines that were distinct for the two lung cancer types. SILA 421 was less cytotoxic against A549 NSCLC cells (IC50 16.63±3.38 M) but proved to have profound effects against SCLC cells (GLC14: IC50 1.75±0.38 M - NCI-H417: IC50 10.63±0.75 M). Significantly higher activity was yielded in GLC14 (IC50 1.75±0.38 M) than in GLC19 (IC50 3.88±1.00 M) SCLC cells, which had both been derived from the same lung cancer patient before and after chemotherapy, respectively. Finally, MTT tests investigating the antiproliferative activity of SILA 409 gave significantly lower cytotoxic activity due to the morpholino substituent on the side chain than the structurally related piperazinosubstituted derivative SILA 421 (data not shown). Effect of SILA 421 on Cell Cycle Distribution in HL-60 and MDA-MB-435 Cells To assess alterations of cell cycle distribution induced by the compound cells were treated with SILA 421 for three days and cell Olszewski-Hamilton et al. cycle distribution measured flow cytometrically following PI staining of the cells (Fig. 4). Incubation of HL-60 cells with the test compound in concentrations 2.5 M led to cell cycle arrest in S phase (Fig. 4, top), with 11.3% (0.31 M SILA 421) and 13.0% (2.50 M SILA 421) more cells accumulated in S phase in comparison to untreated control samples. Similarly, MDA-MB-435 breast cancer cells arrested in S phase (+8.1±0.3%) at concentrations 5 M SILA 421 (Fig. 4, bottom). A highly cytotoxic dose of 10 M SILA 421 led to accumulation of the remaining cells in G1/0 phase and reduction of cells in S and G2/M phase. The proportion of apoptotic subG1 cells was approximately 19-fold increased (data not shown). In conclusion, treatment of the cells with SILA 421 resulted in accumulation of cells in S phase. Apoptotic Activity of SILA 421 Investigation of the type of cell death was carried out in one normal and a panel of cancer cells lines following incubation with 25 M SILA 421 for three days and double-labeling with annexin V and PI by flow cytometric analysis. Fig. (5) demonstrates the proportions of apoptotic or late apoptotic/necrotic cells, respectively, resulting from treatment with SILA 421. The data indicate activation of the cellular machinery that mediates programmed cell death by SILA 421 in a cell-type specific manner. The cell Fig. (4). Alterations of cell cycle distribution induced by treatment of HL-60 promyelocytic leukemia (top) and MDA-MB-435 breast cancer cells (bottom) with SILA 421 at indicated concentrations. Values are presented as mean±SD (n=3). All differences to control cells, except for G2M at 0.625 and 1.25 M SILA 421 for HL-60 and for G2M at 1.25-5 M SILA 421 for MDA-MB-435 cells, respectively, were statistically significant. Anticancer Effects of the Organosilicon Multidrug Resistance Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 0 5 Fig. (5). Proportion of apoptotic cancer cells resulting from treatment with 25 M SILA 421 for three days. Untreated control cells exhibited a viability exceeding 95% (data not shown). Values are presented as mean±SD (n=3). Table 1. P value Cellular Pathways in HL-60 Cells Significantly Upregulated in Response to SILA 421 Identifier of This Event Name of This Event 2.70E-50 REACT_1014 Translation 1.64E-39 REACT_17015 Metabolism of proteins 8.41E-05 REACT_18356 Unfolded Protein Response 0.000182937 REACT_23810 Calnexin/calreticulin cycle 0.000456771 REACT_12484 EGFR downregulation 0.000578497 REACT_13696 NF-B is activated and signals survival 0.000917956 REACT_22208 Transfer of N-glycan to the protein 0.001986288 REACT_1859 Pentose phosphate pathway (hexose monophosphate shunt) 0.003041131 REACT_6826 Degradation of multiubiquitinated cell cycle proteins 0.005209349 REACT_13776 p75 NTR receptor-mediated signaling 0.010335316 REACT_16952 Protein folding 0.010656859 REACT_11061 Signaling by NGF 0.011772147 REACT_11045 Signaling by Wnt 0.012033128 REACT_6828 APC/C-mediated degradation of cell cycle proteins 0.014006561 REACT_17004 Chaperonin-mediated protein folding 0.01523376 REACT_2254 G1/S DNA Damage Checkpoints 0.01523376 REACT_13648 Regulation of Apoptosis 0.016174794 REACT_9417 Signaling by EGFR 0.029840706 REACT_11123 Membrane Trafficking 0.044836273 REACT_14855 Lactate + H+ [extracellular] <=> lactate + H+ [cytosol] Cells were either untreated or incubated with 1.25 M SILA 421 for four days and gene expression was analyzed using Applied Biosystems Human Genome Survey V2.0 microarrays. Genes where transcription was most strikingly altered (cutoff value: > threefold increased gene expression) were subjected to overrepresentation analysis using the Reactome database available at http://www.reactome.org/ to identify the major cellular pathways upregulated by SILA 421. lines showed different proportions of apoptotic and late apoptotic/necrotic cells, which did not correlate with the respective IC50 values. Effect of SILA 421 on Gene Expression in HL-60 Cells In order to assess alterations in gene expression but preserve cell viability, HL-60 cells were treated with a low concentration of 1.25 M SILA 421 for four days. Thereby, 184 gene transcripts were found to be more than threefold upregulated and 333 transcripts were more than threefold downregulated in response to SILA 421 treatment (data not shown). Pathway analysis of these genes was performed using the Reactome software and the most significant results are listed in Tables 1 and 2. 6 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 0 Table 2. Olszewski-Hamilton et al. Cellular Pathways in HL-60 Cells Significantly Downregulated in Response to SILA 421 P value Identifier of This Event Name of This Event 1.71E-25 REACT_152 Cell Cycle, Mitotic 1.47E-18 REACT_383 DNA Replication 1.13E-10 REACT_6769 Activation of ATR in response to replication stress 3.47E-08 REACT_1538 Cell Cycle Checkpoints 4.71E-05 REACT_216 DNA Repair 2.52E-04 REACT_22344 Nucleosome assembly 1.54E-03 REACT_25201 Kinesins 4.17E-03 REACT_1698 Metabolism of nucleotides 4.35E-03 REACT_847 Urea cycle 4.86E-03 REACT_1046 Pyruvate metabolism and Citric Acid (TCA) cycle 5.29E-03 REACT_6828 APC/C-mediated degradation of cell cycle proteins 7.00E-03 REACT_19218 Na+/H+ exchanger transport (at trans-golgi membrane) 3.75E-02 REACT_1006 Polo-like kinase mediated events 4.56E-02 REACT_1788 Transcription Cells were either untreated or incubated with 1.25 M SILA 421 for four days and gene expression was analyzed using Applied Biosystems Human Genome Survey V2.0 microarrays. Genes where transcription was most strikingly altered (cutoff value: > threefold decreased gene expression) were subjected to overrepresentation analysis using the Reactome database available at http://www.reactome.org/ to identify the major cellular pathways downregulated by SILA 421. DISCUSSION MDR in cancer cells is a major cause of failure of anticancer chemotherapy. One of the major determinants of the MDR phenotype is the overexpression of ABC transporters, which include ABCB1 (P-glycoprotein/MDR1), ABCCs (MRP) and ABCG2 (BCRP/MXR/ABCP) [9]. When these transporters are overexpressed in cancer cells, they extrude structurally and mechanistically different chemotherapeutic drugs, thereby lowering intracellular drug concentration below effective levels [10]. The expression of specific ABC transporters is associated with tumorinitiating cells or cancer stem cells in several types of cancer [11, 12]. For more than 30 years, investigators have been making a significant effort to develop specific inhibitors/modulators that can reverse MDR in cancer cells. The first-generation inhibitors/ modulators, such as verapamil and cyclosporine A, showed serious adverse effects at doses required to reverse MDR significantly. Second-generation inhibitors/modulators, such as SDZ PSC833, were found to promote toxicity through pharmacokinetic interference and high affinity third-generation inhibitors/modulators including LY335979, GF120918 and MS-209 lacked significant efficacy in late-phase clinical trials [13, 14]. In view of these results it was clear that there was a need to develop and test efficacious inhibitors/modulators. Two organosilicon compounds, namely SILA 409 and SILA 421, based on a phenothiazine-like structure, were developed by Molnar et al. [2]. Both compounds in the low M range were shown to inhibit efflux of fluorescent dyes in mouse and human cell lines expressing P-gp. The MDR1 gene was not downregulated and the compounds did not exhibit activity against MRP1-mediated transport. In these low concentrations the SILA compounds had an antiproliferative effect, without induction of apoptosis and acted synergistically with epirubicin against an MDR1-overexpressing colon cancer cell line. A further study on variants of MCF-7 breast cancer cell lines that were highly resistant to taxanes, doxorubicin and vinblastine revealed synergistic cytotoxicity for the SILA compounds and paclitaxel, as well as docetaxel [3]. Several other investigations showed a wide spectrum of activities of these organosilicon compounds, such as elimination of drug resistance plasmids from bacteria, vascular activity through modulation of the intracellular Ca2+ concentration, killing of intracellular extensively drug-resistant Mycobacterium tuberculosis strains and downregulation of an endoplasmic reticulum (ER) chaperone involved in resistance to etoposide in a human SCLC cell line [5-7, 15]. These findings indicate that the two SILA compounds, in dependence of the concentrations used, affect cellular mechanisms apart from ABC transporter-mediated drug redistribution. According to our own results, the substituted phenothiazine-like molecule SILA 421 holds significant cytotoxic potential in a broader panel of tumor cell lines that do not overexpress P-pg, while the related compound SILA 409 proved to exert little antiproliferative activity. Highest effects of SILA 421 were found for SCLC and, secondly, gastrointestinal cancer cell lines, whereas untransformed HEK-293 cells were less susceptible. Interestingly, SILA 421 exerted low activity against hormone-insensitive PC-3 prostate cancer cells, whereas proliferation of the hormonesensitive prostate cancer cell line LNCaP was considerably affected. Increased resistance to SILA 421 was observed for succeeding samples from an SCLC patient before and after chemotherapy with doxorubicin/cyclophosphamide/etoposide [16]. Furthermore, SILA 421 induced cell cycle arrest in the S phase in HL-60 and MDA-MB-435 cells and apoptosis or apoptosis/necrosis in a cell-type specific manner. These findings prompted us to investigate the mechanism of action in order to identify putative mediators of the cytotoxic action of SILA 421. HL-60 leukemia cells were treated with this compound and significantly up- and downregulated gene transcripts detected in global gene expression arrays. Protein translation (REACT_1014, REACT_17015) in conjunction with the unfolded protein response (UPR; REACT_ 18356) are two of the most outstanding upregulated pathways in SILA 421-treated HL-60 cells. UPR is activated in response to accumulation of unfolded or misfolded proteins in the lumen of the ER [17]. It aims at restoring normal cellular function first by a halt of protein translation and second by activation of signaling pathways, which lead to an increase of the production of molecular chaperones that assist in the protein folding. In the case these objectives are not achieved within a certain time lapse or the disruption is prolonged, UPR will lead to apoptosis. In addition, Anticancer Effects of the Organosilicon Multidrug Resistance the upregulated calnexin/calreticulin cycle (REACT_23810) is critical for quality control of nascent proteins [18]. The transfer of N-glycan to the proteins (REACT_22208) occurs cotranslationally at the lumenal side of the ER membrane when growing polypeptide strands are translocated from ribosomes into the ER [19]. For example, the glycosylation of the MDR mediator P-gp is important for the functionally active conformation of the drug transporter [20]. Chaperonin-mediated protein folding (REACT_17004) furthermore involves prefoldines, i.e., transfer proteins that bind specifically to cytosolic chaperonin (c-CPN), thereby forming a complex with other nascent proteins to fold these correctly [21]. Moreover, SILA 421 downregulated expression of the epidermal growth factor receptor (EGFR) (REACT_12484), which in particular implicates ubiquitination of stimulated EGFR (Cbl:Grb2; REACT_12562) [22]. p75 NTR receptor-mediated (REACT_13776) and NGF-induced (REACT_11061) signaling both cooperative in activating NF-B and the tumor necrosis factor receptor-associated death domain protein (TRADD) were upregulated, indicating an antiapoptotic activity of SILA 421 [23]. Signal transduction by Wnt (REACT_11045), found upregulated as well, effectuates a series of events mediated by activation of cell-surface receptors of the frizzled family by binding of members of the Wnt family of extracellular ligands. This initiates a signal-transduction cascade that sequentially involves the cytosolic protein Dishevelled and the serine-threonine kinase glycogen synthase kinase GSK-3 and stabilization of -catenin in the cytosol [24-27]. Dishevelled is a main component of a membrane-associated Wnt receptor complex, which, when activated by Wnt binding, inhibits the complex axin/GSK-3/adenomatous polyposis coli (APC) and blocks destruction of -catenin. Thereby -catenin is able to enter the nucleus and to interact with transcription factors of the TCF/LEF family to promote specific gene expression. In line with this and in good agreement with the observed cell cycle arrest induced by SILA 421 our data reveals upregulation of the expression of genes that are involved in APC/C:Cdc20-mediated degradation of mitotic proteins (REACT_6781) [28]. Similarly, expression of genes relevant for activation of the NF-B survival pathway (REACT_13696) was enhanced, which most likely may be caused by ER stress [29]. Increased gene expression of mediators of the pentose phosphate pathway (hexose monophosphate shunt; REACT_1859) pointed to an attempt of the cell to provide NADPH for the maintenance of glutathione levels and reducing cellular conditions [30]. The upregulated monocarboxylate transporter SLC16A3 (REACT_14855) is known to catalyze rapid transport of many monocarboxylates such as lactate, pyruvate, branched-chain oxo acids derived from leucine, valine and isoleucine and the ketone bodies acetoacetate, -hydroxybutyrate and acetate across the plasma membrane. Altogether these upregulated processes are in accordance with cellular compensatory mechanisms that aim at the handling of an increased occurrence of incorrectly translated and misfolded proteins through upregulation of genes involved in translation, UPR, ER quality check and protein folding. Consequently, the observed cell proliferation seems to be reduced by the downregulation of EGFR signaling on the one hand and increased gene expression related to degradation of mitotic proteins on the other hand. NF-B and NGF cooperatively generate antiapoptotic signals, and cellular metabolic pathways are adapted to provide supporting reducing and pH conditions. Further genes that were downregulated in HL-60 cells in response to SILA 421 treatment affected the mitotic cell cycle (REACT_152), cell cycle proteins and cell cycle checkpoints in general (REACT_1538) as well as the metabolism of nucleotides (REACT_1698). Decreased activation of ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) in response to replication stress (REACT_6769), members of the phosphatidylinositol 3kinase related kinase (PIKK) family, impairs their function as Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 0 7 initital DNA damage sensors [31]. Their role is to respond to DNA lesions generated following replication fork stalling in S-phase or UV damage, respectively. ATR, as soon as activated, triggers checkpoint responses by phosphorylation of downstream target proteins, such as Chk1, Chk2 and p53, to provoke cell cycle arrest, DNA repair or apoptosis. In good agreement with these results activation of claspin (REACT_6750) was downregulated. Replication stress and DNA damage trigger an association of claspin with the cell cycle checkpoint regulator Chk1, which is thus activated [32]. A central player during checkpoint recovery inclosed in the genes downregulated by SILA 421 is the Polo-like kinase 1 (Plk1) [31]. Checkpoint recovery (REACT_1006; Polo-like kinase mediated events), another cell cycle-related pathway downregulated in the HL-60 cells, switches off the replication stress and DNA damage-induced cellular events once the damage has been repaired and thus controls the continuation of the cell division process. Transcription of double-strand break repair (REACT_2054) genes like TP53BP1, RAD51, MRE11A, RPA3, NBS1 and BRCA1 was as well reduced, as was the transcription process in general (REACT_1788) [33]. Downregulation of the kinesines motor proteins (REACT_25201) affects several cellular functions such as mitosis, meiosis and transport of cellular cargo, since the kinesins are proteins ATP-dependently sliding along the microtubule filaments [34]. All antimitotic drugs that have been approved so far target the spindle microtubules, which results in mitotic arrest and apoptosis. However, these drugs are also associated with a variety of side effects like neurotoxicity. Due to their specific function in mitosis, targeting of kinesins seems to present an opportunity for development of more selective antimitotics with a superior side-effect profile and activity against cancer cells resistant to microtubule-targeting taxanes. Finally, pyruvate metabolism/ ketone bodies utilization was reduced by SILA 421 (REACT_2071, REACT_59, REACT_1861) [35]. In summary, SILA 421 seems to interfere with the production of correctly expressed proteins indicated by upregulation of UPR and ER stress responses, which in turn appears to lead to further damage to the DNA replication and transcription machinery. Since the resulting defects are expected to exceed the repair capability of the cell, cell cycle arrest is followed by downregulation of doublestrand break repair and replication stress response pathways, consequently leading to (apoptotic) cell death. The design of inhibitors of P-gp still represents a challenging task for medicinal chemists, since its polyspecificity combined with the limited structural information available makes drug design approaches rather ineffective [36]. The two phenothiazine-like SILA compounds possess structural similarity to verapamil and low concentrations were demonstrated to display similar P-gp inhibitory and chemosensitizing activity in drug efflux and cytotoxicity assays, respectively. SILA 409 and SILA 421 fulfil the prerequisites of effective modulators, namely logP value 2.92, a molecular axis 18 atoms, high tendency to release electrons specified by the energy of the highest occupied orbital (Ehomo) value and at least one tertiary basic nitrogen atom [37]. Additionally, insertion of silicon atoms confers increased lipophilicity and stability to the molecule [38]. SILA 421 at higher concentrations, just as well as at lower doses in cell lines with increased sensitivity, exhibits cytostatic and cytotoxic effects, which do not seem to be related to a modulation of P-gp activity or transcription. Thus, the pleotropic effects of SILA 421 comprising antimicrobial toxicity, elimination of resistance plasmids in Escherichia coli and vascular activity need to be explained by cellular mechanisms that are shared with the anticancer effects. Low doses of verapamil in vitro reduced thymidine incorporation into lymphocytes and high doses were lethal for the cells, partially in dependence of the transmembrane Ca2+ flux [39]. This is in good agreement with the ability of the SILA compounds to disturb the intracellular Ca2+ homeostasis in 8 Anti-Cancer Agents in Medicinal Chemistry, 2012, Vol. 12, No. 0 endothelial cells at higher concentrations. Our results indicate that SILA 421 targets some basic mechanisms involved in DNA replication and transcription, resulting in the formation of misformed proteins, cell cycle arrest and downregulation of repair mechanisms, leading to cytostasis and apoptotic cell death eventually. Antimicrobial activity and the elimination of resistance plasmids in Escherichia coli are most likely linked to the SILA 421-sensitive components of the replication machinery shared between mammalian and microbial enzymes. Cell proliferation is a precondition for the cytotoxic action of SILA compounds, since resting lymphocytes showed no decrease of viability after treatment with concentrations 125 M [40]. Furthermore, the compounds were synergistic with paclitaxel and docetaxel in resistant MCF-7 variant cell lines; however, combinations with the P-gp substrate vinblastine proved to be less active. Thus, the synergism with taxanes may be partially due to downregulation of the expression of kinesins and mitotic arrest. Overexpression of kinesin KIFC3 was reported to confer taxane resistance to breast cancer cells and, likewise, overexpression of any of several different kinesins (KIFC3, KIFC1, KIF1A and KIF5A) including both N- and C-kinesins was associated with docetaxel resistance in breast cancer cells [41, 42]. An intensive search for inhibitors of kinesins is ongoing to provide appropriate drugs with the potential to overcome cellular resistance to microtubule-directed taxanes [34, 43]. CONCLUSION SILA 421 has profound effects on cell proliferation and viability, apart from its function as a P-gp modulator. Its anticancer activity affecting DNA replication/transcription and cell cycle arrest in combination with inhibition of mitotic proteins and downregulation of repair mechanisms should therefore be further investigated. Olszewski-Hamilton et al. [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] CONFLICTS OF INTEREST Declared none. 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