Download Anticancer Effects of the Organosilicon Multidrug Resistance

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.
ACKNOWLEDGMENTS
This work was in part supported by The Foundation for Cancer
Research, Szeged, Hungary. The authors declare that they have no
conflict of interest.
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