Accepted Manuscript

Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells Sara A.A. Abdelfatah , Thomas Efferth PII: DOI: Reference:

S0944-7113(15)00021-5 10.1016/j.phymed.2015.01.002 PHYMED 51774

To appear in:

Phytomedicine

Received date: Revised date: Accepted date:

2 November 2014 7 December 2014 12 January 2015

Please cite this article as: Sara A.A. Abdelfatah , Thomas Efferth , Cytotoxicity of the indole alkaloid reserpine from Rauwolfia serpentina against drug-resistant tumor cells, Phytomedicine (2015), doi: 10.1016/j.phymed.2015.01.002

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Flow cytometric doxorubicin uptake

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Reserpine

Molecular docking

Microarrays

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Cytotoxicity of the indole alkaloid reserpine from Rauwolfia

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serpentina against drug-resistant tumor cells

Sara A. A. Abdelfataha, Thomas Effertha,*

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Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry,

Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany

* Corresponding author:

Tel: 49-6131-3925751; Fax: 49-6131-3923752 E-mail address: [email protected]

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Running title: Reserpine to treat drug-resistant tumors

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Abbreviations: ABC, ATP-binding cassette BCRP, Breast cancer resistance protein EGFR, Epidermal growth factor receptor

MDR, Multidrug resistance

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MAPK, Mitogen-activated protein kinase

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MSH2, MutS homologue 2, mismatch repair enzyme mTOR, Mammalian target of rapamycin NCI, National Cancer Institute PI3K, Phosphoinositid-3-kinase

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STAT3, Signal transducer and activator of transcription 3

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Abstract

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Background: The antihypertensive reserpine is an indole alkaloid from Rauwolfia serpentina and exerts also profound activity against cancer cells in vitro and in vivo. The present investigation was undertaken to investigate possible modes of action to

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explain its activity towards drug-resistant tumor cells.

Material and methods: Sensitive and drug-resistant tumor cell lines overexpressing

P-glycoprotein (ABCB1/MDR1), breast cancer resistance protein (ABCG2/BCRP), or mutation-activated epidermal growth factor receptor (EGFR), wild-type and p53knockout cells as well as the NCI panel of cell lines from different tumor origin were

analyzed. Reserpine’s cytotoxicity was investigated by resazurin and sulforhodamine assays, flow cytometry, and COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based RNA expressions.

Results: P-glycoprotein- or BCRP overexpressing tumor cells did not reveal crossresistance to reserpine. EGFR-overexpressing cells were cross-resistant and p53knockout cells collateral sensitive to this drug compared to their wild-type parental

cell lines. Reserpine increased the uptake of doxorubicin in P-glycoprotein-

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overexpressing cells, indicating that reserpine inhibited the efflux function of Pglycoprotein. Using molecular docking, we found that reserpine bound with even higher binding energy to P-glycoprotein and EGFR than the control drugs verapamil (P-glycoprotein inhibitor) and erlotinib (EGFR inhibitor). Cell cycle analysis showed that reserpine induced G1 phase arrest. COMPARE and cluster analyses of microarray

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data showed that the mRNA expression of a panel of genes predicted the sensitivity or resistance of the NCI tumor cell line panel with statistical significance. The genes belonged to diverse pathways and biological functions, e.g. cell survival and apoptosis, EGFR activation, regulation of angiogenesis, cell mobility, cell adhesion, immunological functions, mTOR signaling, and Wnt signaling.

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Conclusion: The lack of cross-resistance to most resistance mechanisms and the collateral sensitivity in EGFR-transfectants compared to wild-type cells speak for a promising role of reserpine in cancer chemotherapy. Reserpine deserves further consideration for cancer therapy in the clinical setting.

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Keywords: ABC-transporter, Apocynaceae, Cluster analysis, Collateral sensitivity, Molecular docking, Pharmacogenomics 3

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Introduction

Reserpine is an indole alkaloid derived from the roots of Rauwolfia serpentina and serves as potent antihypertensive drug (Panda et al. 2012) It represents an established

second line treatment against hypertension (Milne & Pinkney-Atkinson 2004; Pillay

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2009).

Reserpine mediates the depletion of neurotransmitters from postganglionic nerve endings, which consequently lower arterial pressure and total peripheral resistance ultimately leading to decreased heart rates and cardiac output. Its administration together with diuretics effectively lowers arterial pressure and significantly reduces morbidity and mortality related to hypertension (Bakris & Frohlich 1989).

Early studies started at the mid-1950s reported anti-tumor effect of reserpine in vivo independent from its cardiovascular action. Experimental studies in mice bearing advanced leukemia, reserpine increased animals’ life span by three-fold (Burton et al.

1956). Other studies reported the anti-tumor activity of reserpine in different mouse sarcomas in vivo (Belkin & Hardy 1957; Nelson et al. 1981).

Since the 1980s, attention was paid towards phenomena related to chemotherapy

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failure (Efferth 2001; Gillet et al. 2007; Eichhorn & Efferth 2012) and the ATP

binding cassette (ABC) transporter, P-glycoprotein (ABCB1), which confers multidrug resistance (MDR) by energy-dependent drug efflux process (Hall et al. 2009). Drugs such as verapamil, quinidine, tamoxifen, progesterone, rapamycin, cyclosporins and others were found to inhibit P-glycoprotein and to overcome MDR

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(Arceci 1993). Interestingly, reserpine was also reported as P-glycoprotein inhibitor. It suppressed photolabeling of P-glycoprotein with a vinblastine analogue in MDR cell lines (Akiyama et al. 1988). Reserpine and other indole alkaloids enhanced the sensitivity of MDR cancer cells towards cytotoxic agents (Beck et al. 1988; Zamora et al. 1988).

In this study, we investigated the role of reserpine in different cancer cell lines in an

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approach to understand possible molecular modes of action. Since MDR is multifactorial in nature and other mechanisms in addition to P-glycoprotein also contribute to unresponsiveness of tumors, we also investigated several other mechanisms. The ABC-transporter BCRP/ABCG2, the mutated tumor suppressor gene p53 and the activated epidermal growth factor receptor (EGFR) all mediate drug

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resistance (el-Deiry 1997; El-Deiry 2003; Gillet et al. 2007). Therefore, we 4

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investigated, whether or not cell lines expressing these genes reveal cross-resistance to reserpine. The intention was to prove, whether reserpine could be used to bypass

Material and Methods Cell Culture

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drug-resistance and to eradicate otherwise unresponsive tumors by reserpine.

Drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine

serum (FBS) (Invitrogen) and 1% penicillin (100 U/ml)-streptomycin (100 μg/ml) (PIS) antibiotic (Invitrogen) and incubated in humidified 5% CO2 atmosphere at 37 °C.

Breast cancer cells transduced with control vector (MDA-MB-231-pcDNA3) or with

a cDNA for the breast cancer resistance protein BCRP (MDA-MB-231BCRP clone 23), human wild-type HCT116 (p53+/+) colon cancer cells as well as knockout clones HCT116 (p53−/−) derived by homologous recombination, non-transduced human

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U87MG glioblastoma multiforme cells and U87MG cells transduced with an

expression vector harbouring an epidermal growth factor receptor (EGFR) gene with a genomic deletion of exons 2 through 7 (U87MG.ΔEGFR) were all maintained in DMEM medium, supplemented with 10% FBS and 1% penicillin-streptomycin and incubated under standard conditions as described for leukemia cell lines.

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The resistance of the different resistant cell lines has been maintained by using 5000 ng/ml doxorubicin for CEM/ADR5000, 400 μg/ml geneticin for U87MG.ΔEGFR and HCT116 (p53−/−) and 800 ng/ml of the same compound for MDA-MB-231 BCRP clone 23.

The glioblastoma cell lines were kindly provided by Dr. W. K. Cavenee (Ludwig Institute for Cancer Research, San Diego, CA). Transfected breast cancer cell lines

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were a generous gift from Dr. B. Vogelstein and H. Hermeking (Howard Hughes Medical Institute, Baltimore, MD). The leukemia cells were kindly provided by Dr. J. Beck (Department of Pediatrics, University of Greifswald, Greifswald, Germany).

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Cytotoxicity Assay

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The cytotoxicity of reserpine has been investigated using the resazurin reduction

assay (Borra et al. 2009). Resazurin is an indicator dye, which is reduced in viable cells to highly fluorescent resorufin, in contrast to non-viable cells, which lost their metabolic capability and are not able reduce resazurin. 96-well cell culture plate (Thermo Scientific, Germany) were seeded with 20,000 cells/well in a total volume of

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100 μL, and then treated with different concentrations of reserpine diluted in 100 μL

medium. Adherently growing cells were allowed to attach overnight and treated after 24 h. The cells were incubated with reserpine for 72 h. Then, 0.01% of resazurin

(Sigma-Aldrich, Germany) diluted in double distilled water (ddH2O) was added (20 μl/well) and incubated for another 4 h. Infinite M2000 Pro™ plate reader (Tecan,

Germany) was used to measure the fluorescence using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Experiments were performed three

times with at least with six replicates per experiment. The 50% inhibition concentrations (IC50) were calculated from dose response curves of each cell using Microsoft Excel 2013 software.

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Doxorubicin uptake assay

Flow cytometry has been used to measure the retention of doxorubicin. Doxorubicin is substrate of P-glycoprotein and its inherent fluorescence was used to assess the efflux activity of this drug transporter. CCRF-CEM and CEM/ADR5000 cells were seeded in phenol red–free RPMI 1640 medium in a concentration of 5×105 cells/well in 12 well plates at a total volume 500 μl. Then, cells were treated with medium

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containing 10 μM doxorubicin with and without reserpine (15 μM). Cells were incubated at 37 °C in an atmosphere containing 5% CO2 for 3 h, which is the time required for maximum doxorubicin uptake (Krishan & Hamelik n.d.). Finally, cells were washed to remove free doxorubicin. Cells were measured on LSR-Fortessa FACS analyzer (Becton-Dickinson, Germany) equipped with argon blue laser, the

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excitation and emission wavelength of doxorubicin were 488 nm and 610/20 nm, respectively. Only living cells, identified by DAPI to stain dead cells were considered for the analyses. Data were processed by Flowjo software. Three controls were taken, unstained cells to determine auto-fluorescence, cells treated with doxorubicin alone to measure the efflux efficacy, and in combination with verapamil as positive control for

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a P-glycoprotein inhibitor. 6

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Molecular Docking

AutoDock4 (Hetenyi & Van Der Spoel 2002; Morris et al., 2009) was used for molecular docking calculations of reserpine. A homology model human ABCB1

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based on the crystal structure of murine P-glycoprotein was previously constructed by us (Zeino, 2014; (Tajima et al. 2014) and used in the present investigation for binding site determination of reserpine. Verapamil was included in our analyses as control drug for a well-known P-glycoprotein inhibitor.

Molecular docking of reserpine to EGFR was done on the ATP-binding site of EGFR kinase domain. The crystal structure of EGFR was retrieved from The Protein Data BANK

(Database

code

PDB1M17;

(http://www.rcsb.org/pdb/home/home.do).

Erlotinib which is an irreversible tyrosine kinase inhibitor of EGRF was taken as

control drug for docking. The 3D structure of reserpine, verapamil and erlotinib were downloaded from PubChem (pubchem.ncbi.nlm.nih.gov).

Drug binding residues of ABCB1 were identified as His61, Gly64, Leu65, Met69,

Ser222, Leu304, Ile306,Tyr307, Phe336, Leu339, Ile340, Ala342, Phe343, Gln725,

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Phe728,Phe732, Leu762, Thr837, Ile868, Gly872, Phe942, Thr945, Tyr953,Leu975,

Phe978, Ser979, Val982, Gly984, Ala985, Met986, Gly989,Gln990, and Ser993 (Aller et al. 2009). At the other hand, residues identified as binding sites for EGFR include Leu620, Leu694, Phe699, Val702, Ala719, Lys721, Met742, Leu764, Thr766, Gln767, Met769, Pro770, Cys773, Thr830 and Asp831 (Yadav et al. 2014).

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Crude BDP structures of the receptors proteins were refined, energy minimized and polar hydrogens were added and saved as pdbqt files. Then, the grid map parameters were set to cover the defined residues. Numbers of runs and energy evaluations were reset to 250 and 25,000,000, respectively. Docking calculations were performed using Lamarckian Genetic Algorithm. For image visualization of docking results, Visual

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Molecular Dynamics (VMD) software was used.

COMPARE and hierarchical cluster analyses of microarray data. Messenger RNA expression profiles of 60 human cancer cell lines were deposited at the database of the Developmental Therapeutics Program (DTP) of the National

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Cancer Institute (NCI) (http://dtp.nci.nih.gov).

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Compare analysis (Paull et al. 1989) was performed to correlate IC50 values for reserpine and microarray-based transcriptome-wide mRNA expression levels in the NCI cell line panel. According to gene expression levels, Resistance and sensitive

candidate genes were determined using standard and reverse COMPARE as described

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(Villeneuve & Parissenti 2004; Zeeberg et al. 2011; Reinhold et al. 2012)

Pearson’s rank correlation test was used (WINSTAT program, Kalmia) for calculation

of significance values (p-values) and ranking correlation coefficients (R-values) as a

relative measure of the linear dependency of two variables. The median log10 IC50 value was taken as a cut-off threshold for determination of cell lines being sensitive or resistant to reserpine.

Hierarchical cluster analysis (WARD method) groups objects into clusters according to similarities and closeness between them. For calculation of distances of all

variables involved in the analysis, the program automatically standardizes the variables by transforming the data with a mean = 0 and a variance = 1. Then, cluster

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trees were performed.

Results

Resazurin reduction assay

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The cytotoxic effects of reserpine have been investigated in different cancer cell lines (Fig. 1) to gain insight into the mechanisms of action underlying the activity of the compound. Reserpine exerted 50% cell viability inhibition in parental CCRF-CEM cells at a concentration of 14.52 ± 1.62 µM and of 13.2 ±1.02 µM in P-glycoproteinoverexpressing, multidrug-resistant CEM/ADR5000 cells. These results clearly show that multidrug-resistant cells did not exhibit cross-resistance to reserpine, although

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these cells are highly resistant to established anticancer drugs such as anthracyclines, Vinca alkaloids, taxanes, epipodophyllotoxins and others (Efferth et al. 2008). Treatment

of

drug-sensitive

MDA-MB-231-pcDNA3

and

BRCP-transfected,

multidrug-resistant MDA-MB-231-BCRP (clone 23) resulted in IC50 values of 34.33

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± 10.38 µM and 40.6 ± 6.84 µM, respectively.

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U87MG cells transfected with mutation-activated EGFR (U87MG.ΔEGFR) were

much more sensitive towards reserpine (IC50: 9.15 ± 2.67 µM) than wild-type cells

(87.98 ± 11.84 µM), indicating that EGFR-overexpressing cells display collateral sensitivity (hypersensitivity) to reserpine.

Finally, we compared the responsiveness of p53 wild-type and knockout cells towards

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reserpine. HCT116 (p53+/+) colon cancer cells were sensitive to reserpine (IC50: 30.07 ± 7.57 µM), while HCT116 (p53−/−) cells were resistant to reserpine.

Doxorubicin uptake assay

Doxorubicin uptake was measured in terms of fluorescence intensity, which can be

taken as a measure for intracellular accumulation of the drug. CCRF-CEM and Pglycoprotein-overexpressing CEM/ADR5000 cells were both incubated with

doxorubicin with or without reserpine. CCRF-CEM cells that do not express Pglycoprotein were sensitive to doxorubicin and neither reserpine nor the control drug verapamil

shows

any

effect

on

doxorubicin

accumulation.

By

contrast,

CEM/ADR5000 cells showed only intracellular low fluorescence intensity of

doxorubicin (Fig. 2), The fluorescence intensity of doxorubicin considerably

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increased after addition of 3.75 μM reserpine (0.25×IC50 value) and even more increased after incubation with 15 μM reserpine (IC50 value). The retention effect of reserpine (IC50 value) was 2.04-fold higher than of verapamil. It is important to note that there were no auto-fluorescent effects from either tested cancer cell lines.

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Molecular docking of reserpine to ABCB1 and EGFR Reserpine showed a high binding energy for human P-glycoprotein/ABCB1 (-9.65 ± 0.79 kcal/mol), even higher than that of the control drug verapamil (8.57  0.13 kcal/mol) (Table 1). Two hydrogen bonds with drug binding residues (Gly226 and Lys234) interacted with reserpine. Interestingly, reserpine bound to the same drug binding site as verapamil (Fig. 3A). The residues involved in this interaction were

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Ser222, Pro223, Gly226, Ala229, Ala230, Lys234, Phe 303, Ile306, Tyr310, Leu339, Ala342, Phe 343 and Gly 346. Molecular docking of reserpine to the tyrosine kinase domain of EGFR (PDB IM17) revealed a low binding energy (-7.32 ± 1.12 kcal/mol). Two hydrogen bonds were

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involved in this interaction (Lys721 and Met769). It is remarkable that reserpine

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revealed a higher binding affinity than the control EGFR inhibitor, erlotinib (-5.93 ± 0.3 kcal/mol). Both reserpine and erlotinib shared the same residues involved in

hydrophobic interaction (Fig. 3B), i.e. Leu694, Lys721, Glu738, Leu768, Met769,

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Gly772, Cys773, Asp813, Arg817, Leu820 and Asp831.

Cross-resistance of reserpine to established anticancer drugs

To get a clue on possible modes of actions of reserpine, we correlated the log10IC50 values of the NCl cell lines to reserpine with those of 87 standard drugs. The cellular

responses of 4 out of 7 anti-hormonal drugs significantly correlated with those of reserpine (= 57%). Alkylating drugs were also frequently correlated to reserpine.

Seven of 13 alkylating agents (= 54%) revealed significant correlations to reserpine (p < 0.05; R > 0.30). Comparable results were obtained for mTOR inhibitors (2/4 drugs

= 50%). Intermediate correlation rates were found for DNA topoisomerase inhibitors

(3/8 drugs = 37.5%) antimetabolites (2/15 drugs = 13%), as well as tyrosine kinase inhibitors (1/13 drugs = 8%). No correlations were found for platin compounds,

tubulin inhibitors, and epigenetic inhibitors (Fig. 4). These results may indicate that

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reserpine exerts multiple modes of action, a feature which is frequently observed with phytochemicals (Efferth and Koch, 2011).

COMPARE and cluster analyses of microarray data

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COMPARE and cluster analyses of microarray data have shown that cell lines of different tumor types show different degrees of sensitivity to reserpine (Fig. 5). In an approach

to

responsiveness

identify molecular pharmacology and mechanism to

reserpine,

candidate

genes

underlying

have been identified using

transcriptome-wide COMPARE analysis. Using Pearson test, genes were ranked whose mRNA expression directly or inversely correlate with the log10IC50 values for

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reserpine of all NCI cell lines (Table 2). Only genes with correlation coefficients of R > 0.5 and R < −0.5 were considered for direct inverse correlations respectively. These genes belonged to pathways and biological functions that presumably determined responsiveness of tumor cells to reserpine, e.g. cell survival and apoptosis

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(TRAF1, SULF1), EGFR activation (EFEMP1, EPS15L1), regulation of angiogenesis 10

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(ANGPTL4), cell mobility (ACTA2, HSPB3, CNN1), cell adhesion (POSTN, MOG),

immunological function (CFI, LBP, IL17B), mTOR signaling (PPAPDC3) and Wnt signaling pathway (DIXDC1, SFRP2, CPZ, ZRANB1).

Hierarchical cluster analysis was performed using the mRNA expression values listed

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in Table 2. As shown in Fig. 6, the resulting dendrogram could be separated into four main branches. The median value of the log10IC50 values of reserpine was then used as cutoff value to define cell lines as being sensitive or resistant to reserpine. The

distribution of sensitive and resistant cell lines is shown in Table 3. Interestingly, a statistically significant distribution pattern across the four dendrogram branches was

observed. The majority of cell lines in clusters 1 and 2 were reserpine-resistant,

whereas those in clusters 3 and 4 were mainly sensitive (P=0.00437; chi2 test). Since only mRNA values but not log10IC50 values were included into the cluster analysis, the mRNA expression profile alone was sufficient to predict sensitivity or resistance

to reserpine. Cluster 1 and 2 contain both sensitive and resistant cell lines to reserpine

Discussion

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whereas cluster 3 and 4 contain only resistant cell lines to reserpine.

Cross-resistance and collateral sensitivity of drug-resistant cell lines

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Our results showed that reserpine has novel cytotoxic features that may be beneficial for the treatment of drug-resistant tumors. P-glycoprotein-overexpressing cells were not cross-resistant to reserpine, indicating that this compound may be valuable to eradicate MDR tumor cell populations of refractory tumors. The early reports from the 1950s on the anticancer activity of reserpine in vivo (Burton et al. 1956; Belkin & Hardy 1957) did not went into further mode of action analyses, since major molecular

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mechanisms were still unknown at that time. For instance, apoptosis as important mechanisms of cell death (Kerr et al., 1972) and P-glycoprotein as mediator of multidrug resistance (Juliano and Ling, 1976) came only up in the 1970s. Later on, it has been indeed demonstrated that reserpine triggers apoptotic cell death, which explains the anticancer activity of this drug independent of its calcium channel

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blocking, antihypertensive effects. Reserpine induces cell death by activation of the 11

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TRAIL-mediated apoptotic pathway leading to up-regulation of BAX, downregulation of BCL-2 as well as activation of caspase-3 and caspase-8 (Cantarella et

al., 2009). In the present investigation, we found that reserpine reversed doxorubicin resistance of P-glycoprotein overexpressing CEM/ADR5000 cells. Reserpine is an effective modulator for P-glycoprotein, which inhibits intracellular doxorubicin

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accumulation by binding with high affinity to this ABC-transporters leading to efficient drug efflux inhibition. These results are in accord to previous data reporting

on the photoaffinity labeling of P-glycoprotein and its inhibition by reserpine (Qian & Beck 1990). The inhibition of P-glycoprotein’s efflux function by reserpine was subsequently confirmed by other authors (Schlemmer and Sirotnak, 1994; Bhat et al., 1995; Jetté et al., 1995; Sarver et al., 2002). In addition to these studies, we did not

only confirm reserpine’s inhibitory activity on P-glycoprotein’s function, but we also suggested the amino acids responsible for reserpine’s binding in the pharmacophore

of P-glycopotein by molecular docking. Taken all these results together, reserpine can be understood as a “two-in-one” drug. It kills tumor cells due to its profound

cytotoxicity, and at the same time it inhibits P-glycoprotein’s efflux enhancing the activity of standard drugs such as doxorubicin.

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It was pleasing to observe BCRP-transfectant tumor cells did also not exert crossresistance to reserpine. BCRP (ABCG2) represents another ABC transporter that confers resistance to a broad spectrum of anticancer drugs in various tumor types, including human breast carcinoma, colon carcinoma, gastric carcinoma, fibrosarcoma, and myeloma (Doyle & Ross 2003). The fact that many of the established anticancer

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drugs clinically fail, because of the activity of ABC-transporters such as Pglycoprotein and BCRP illustrates the necessity to identify and develop novel anticancer drugs. Our data give reason to hope that reserpine is a promising candidate drug to improve the effectiveness of cancer chemotherapy. In this context, it was interesting to observe that reserpine was hypersensitive (collateral sensitive) to tumor cells transfected with a mutation-activated form of the

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epidermal growth factor receptor (EGFR). This is an important oncogene, which leads not only to carcinogenesis, but also to tumor progression, metastasis and worse prognosis for survival time of cancer patients (Chanprapaph et al. 2014). EGFR modulates cell survival, proliferation, angiogenesis and migration through activation

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of PI3K, MAPK and STAT3 signaling pathways (Taylor, 2012). EGFR overexpression has been found in several tumor types, including glioblastoma, 12

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colorectal cancer, head and neck squamous cell carcinoma, non-small cell lung

cancer, breast, renal, ovarian, bladder, prostate and pancreatic cancers (Gomez et al. 2013). EGFR overexpression also confers resistance to anticancer drugs (Wykosky et

al. 2011). In the present investigation, we were able to identify a potential novel role for reserpine. The results that EGFR-transfected cells were collateral sensitive to this

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drug compared to non-transfected control cells and that reserpine binds with even

higher affinity than erlotinib to the tyrosine kinase domain of EGFR indicates that reserpine may act as EGFR kinase inhibitor. This hypothesis warrants further more detailed investigations in the future.

Another important determinant of anticancer drug resistance is the tumor suppressor

gene p53. Mutations in p53 lead to functional inactivation causing deregulation of cell

cycle arrest, DNA repair, and apoptosis induction (Liao et al. 2014) . Our results with p53-knockout tumor cells showed that a loss of p53 function was associated with decreased cytotoxicity towards reserpine compared to wild-type p53-proficient cells.

Cross-resistance pattern of the NCI cell line panel between reserpine and standard

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drugs

We performed a large correlation analysis between the log10IC50 values of reserpine and those of 87 standard anticancer drugs with the aim to identify possible modes of action of reserpine. Interestingly, the highest correlation rates were found to antihormonal drugs (57%) and DNA alkylating agents (53%) followed by mTOR

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inhibitors (50%). These results are supported by hints from the literature. Reserpine has previously been investigated in vivo endocrinic disruptor screening assay and was found to interact with the estrogen (Ohta et al. 2012; Koch et al. 1980) reported chemopreventive effects of reserpine. Prolonged estrogen exposure induce pituitary tumors. The authors found that elevated serum prolactin levels in rats induced by ethinyl estradiol were slightly reduced by reserpine.

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Alkylating agents kill cancer cells by alkylation of DNA and subsequent induction of apoptosis. The interaction of reserpine to binds to the DNA repair enzyme MSH2, thereby triggering the MSH2-dependent cell-death pathway (Vasilyeva et al. 2009). In

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this context it is important to mention that despite the inhibition of DNA repair,

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reserpine did not induce genetic damage and was not genotoxic (Tsutsui et al. 1994; Kevekordes et al. 1999) indicating that reserpine may not be carcinogenic.

The correlation of reserpine to two out of four mTOR inhibitors (sirolimus, temsirolimus) is a novel find and has not been recognized before. The hypothetical

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inhibitory effects of reserpine on estrogen receptor and related downstream signaling pathways deserve more detailed investigation in the future.

COMPARE and cluster analyses of microarray data

Apart from the low-level cross-resistance of HCT-116 p53-/- cells to reserpine

compared to wild-type HCT-116 p53+/+ cell, the pother drug-resistant cell lines overexpressing ABCB1, ABCG2, or EGFR did not reveal cross-resistance to

reserpine. This indicates that the responsiveness of tumor cells towards reserpine may be determined by other factors. For this reason, we applied COMPARE and

hierarchical cluster analyses of microarray-based transcriptome-wide mRNA expressions to find out, whether the expression of other than the classical drug

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resistance genes mentioned above correlate with reserpine resistance among the NCI

cell line panel. COMPARE analysis has been reported as valuable method to identify the mode of action of anticancer drugs using the NCI tumor panel (Wosikowski et al. 2000; Evans et al. 2008; Fagan et al. 2012; Luzina & Popov 2012). Furthermore, microarray hybridization and clustering techniques have been widely applied for

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mechanistic studies of established and novel drugs in cancer research (for instance see Reinhold et al., 2012; Villeneuve & Parissenti, 2004; Zeeberg et al., 2011) COMPARE analysis revealed genes belonging to different functional classes, whose mRNA expression correlated to log10IC50 values of reserpine. Remarkably, genes involved in EGFR activation were identified (EFEMP1, EPS15L1). This result and the fact that U87MG EGFR transfected cells were collateral sensitive towards

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reserpine compared to non-transfectant wild-type U87MG cells indicate that EGFR signaling may indeed influence reserpine’s cytotoxicity towards tumor cells. Further experiments shall analyze the signaling processes of EGFR upon reserpine treatment. Furthermore, mRNA expression of genes involved in the regulation of cell survival

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and apoptosis (TRAF1, SULF1) and angiogenesis (ANGPTL4) were observed to 14

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correlate to cellular responsiveness to reserpine. Both apoptosis induction and the formation of new blood vessels in tumors are important factors determining tumor

survival and growth. Therefore, apoptosis-inducing and anti-angiogenic drugs became

effective weapons in the fight against cancer (Efferth 2010; Wahl et al. 2011; Cheng

et al. 2012; Zihlif et al. 2012; Krusche et al. 2013; Seo et al. 2013). Our data indicate

vessel formation.

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that reserpine kills tumors by induction of apoptosis and inhibition of tumor blood

One gene involved in mTOR signaling appeared in our analysis (PPAPD3). Since

reserpine was also correlated with the activity of mTOR inhibitors (sirolimus, temsirolimus), it can be hypothesized that reserpine might disturb mTOR-related signaling routes. Furthermore, the expression of a number of genes involved in cell

mobility (ACTA2, HSP3, CNN1), cell adhesion (POSTN, MOG) and immunological

functions (CFI, LBP, IL17B) appeared in the COMPARE analysis. These biological

functions are all involved in the regulation of tumor microenvironment and metastasis, indicating that reserpine might inhibit metastatic spread of tumors.

In this context, the WNT signaling pathway is important not only for metastasis, but

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also for cancer stem-like cells (Efferth 2012). Whether reserpine reveals cytotoxicity towards stem cells warrants further investigations.

The fact that reserpine that revealed less profound cytotoxicity towards Pglycoprotein-, BCRP- or EGFR-expressing but p53-mutated tumor cells raises the

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question, whether or not these favourable results might be translatable to the clinical situation. Reserpine has been repeatedly shown to inhibit tumor growth in mice (Nelson et al. 1981), indicating that reserpine might not only exert anticancer activity not only in vitro and in animals, but possibly also in human cancer patients. Furthermore, reserpine has been clinically applied for decades to treat cardiovascular diseases. Therefore, using reserpine for cancer therapy as novel indication should not

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be out of reach.

Conclusion and perspectives The anticancer activity of reserpine in vivo is known since the 1950s (Burton et al.

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1956; Belkin & Hardy 1957). However, reserpine had not been further developed as

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cancer drug, probably due to high toxicity. The effects of reserpine on the cardiovascular system may appear as severe side effects in normotonic cancer patients, but may even be beneficial in comorbid cancer patients that suffer from

hypertonia. Furthermore, reserpine may serve as lead compound to develop novel

cardiovascular system.

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derivatives with cytotoxicity against drug-resistant tumors, but without activity on the

Speculating that reserpine would make its way into clinical oncology, it will be used

as part of combination therapy regimens rather than as monotherapy. In this context, the inhibition of P-glycoprotein and increase of intracellular drug accumulation is a

pleasing feature leading to improved cancer killing efficiencies. Previously, it has been reported that reserpine does not only improve the activity of anticancer drugs involved in MDR, but also of alkylating drugs probably by interfering with DNA repair mechanisms (Wakusawa et al. 1984).

Although the antihypertensive reserpine has already been described in the 1950s to

reveal anticancer activity in vivo, its true potential has not been recognized at that time to our point of view. In the present investigation, we reinvestigated this drug

tumors.

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because it reveals surprisingly profound activity towards otherwise drug-resistant

Cancer patients, preferentially older ones, suffer not only from their tumor but also from high blood pressure. Together with the activity of reserpine against drugresistant tumor cells, reserpine might be a valuable supplement to combination

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therapy protocols to treat comorbid patients with cancer and hypertension. In conclusion, the results of the present investigation speak for a promising role of reserpine in cancer chemotherapy due to its activity towards drug-resistant tumor cells. Reserpine deserves reconsideration and re-evaluation for cancer therapy in the

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clinical setting.

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Fig. 1. Cytotoxicity of reserpine towards (A) drug-sensitive CCRF-CEM and Pglycoprotein-expressing, multidrug-resistant CEM/ADR5000 leukemia cell lines, (B) sensitive (MDA-MB-231-pcDNA3) and BCRP-transfected, multidrug-resistant MDA-MB-231-BCRP (clone 23) breast cancer cell lines, (C) sensitive U87MG glioblastoma multiforme cells and EGFR-transfected U87MG.ΔEGFR cells. (D) Human wild-type HCT116 (p53+/+) and knockout HCT116 (p53−/−) colon cancer cells.

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Fig. 2. Flow cytometric histograms of doxorubin uptake in CEM/ADR5000 and CCRF-CEM cells. (a) Untreated control cells (autofluorescence); cells treated with (b) doxorubicin (10 μM), (c) doxorubicin (10 μM) plus verapamil (10 μM), (d) doxorubicin (10 μM) plus reserpine (3.75 μM), and (e) doxorubicin (10 μM) plus reserpine (15 μM).

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Fig. 3. Molecular docking of reserpine to (A) homology modelled human Pglycoprotein/ABCB1 (structures of reserpine in red and of verapamil in yellow) (B) EGFR kinase domain (structure of erlotinib in green)

Fig. 4. Oncobiograms for reserpine using the NCl cell line panel. (A) Percentage of classes of established anticancer drugs, whose log10IC50 values correlate with those for reserpine. (B) Profile of correlation values (R) of anti-hormonal drugs with reserpine. (C) Profile of correlation values (R) of alkylating drugs with reserpine. The log10IC50 values are deposited at the NCl database (http://dtp.nci.nih.gov). The calculations were performed using the Pearson correlation test.

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Fig. 5. Chemical structure of reserpine and mean values and standard deviations of log10IC50 values for reserpine of 43 NCI cell lines of different tumor types.

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Fig. 6. Hierarchical cluster analysis of microarray-based mRNA expression of resistance and sensitive genes obtained by standard and reverse COMPARE analyses. The dendrogram shows the clustering of the NCI cell line panel and indicates the degrees of closeness between cell lines.

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Table 1. Molecular docking of reserpine to P-glycoprotein/ABCB1 and tyrosine kinase domain of EFGR.

ABCB1

EGFR

Compoun d Reserpin e Verapami l Reserpin e Erlotinib

Lowest binding energy (kcal/mol) -9.65 (±0.34) -8.57 (±0.13) -7.86 (±1.57) -6.81 (±0.18)

Mean binding energy (kcal/mol) -9.12 (±0.79) -8.57 (±0.12) -7.32 (±1.12) -5.93 (±0.3)

Residues involved in hydrogen bond Gly226 Lys234

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Target protein

Number of residues involved in hydrophob ic interaction

Lys234 Lys721 Met769 Met769 Cys773

14

pKi*(nM)

10

92.75 (±42.2) 526.79 (±117.04) 531.2 (±361.08)

13

10230 (±0.34)

15

*pKi: predicted inhibitory constant.

Table 2. COMPARE analysis of genes, whose microarray-based mRNA expression correlated with log10IC50 values for reserpine in a panel of 43 cell lines. R-value

Gene Symbol

Genbank Acc. No.

Gene Name

Gene function

MYBPH

NM_0049 97

GC1836 40

Myosin binding protein H

Structural constituent of muscle

0.651

SLC9A9

AI536067

GC6669 6

Solute carrier family 9

Solute hydrogen antiporter activity

0.641

OLFML2B

AL050137

GC8301 3

Olfactomedinlike 2B RNA

Extracellular matrix binding

0.636

BBS9

AF095771

GC1526 97

Bardet-Biedl syndrome 9

Required for ciliogenesis

0.631

EFEMP1

AI740711

GC1590 25

EGF-containing fibulin-like extracellular matrix protein 1

epidermal growth factor-activated receptor activity

0.621

CPZ

U83411

GC9776

Carboxypeptida

Metallocarboxypepti

ED

0.666

PT CE AC

Pattern ID

M

Standard compare (resistan ce genes)

23

se Z

dase activity

TRAF1

U19261

GC3273 9

TNF receptorassociated factor 1

Regulation of cell survival and apoptosis.

0.617

FMO1

NM_0020 21

GC1814 37

Flavin containing monooxygenase 1

N,N-dimethylaniline monooxygenase activity

0.617

TMEM132B

AI435595

GC1570 72

Transmembrane Molecular function protein 132B

0.615

CTNNA2

M94151

GC3481 8

Catenin (cadherinassociated protein), alpha 2

Structural constituent of cytoskeleton

0.614

ANGPTL4

AF169312

GC1535 04

Angiopoietinlike 4

Protein with hypoxiainduced expression in endothelial cells acts as a regulator of angiogenesis

0.613

SULF1

AI479175

GC1572 95

Sulfatase 1

Diminishes proliferation, and facilitates apoptosis

0.611

CFI

Y00318

GC1012 00

Complement factor I

Inactivates complement subcomponents

0.607

CH25H

AF059214

GC5609 2

Cholesterol 25hydroxylase

Cholesterol 25hydroxylase activity

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0 0.618

HSPB3

NM_0063 08

GC1845 72

Heat shock 27kDa protein 3

Inhibitor of actin polymerization

0.603

ACTA2

X13839

GC3574 1

Actin,  2, smooth muscle, aorta

Cell motility

0.602

DIXDC1

AF070621

GC2693 0

DIX domain containing 1

Positive effector of the Wnt signaling pathway

SFRP2

AI246042

GC6148 8

Secreted frizzled-related protein 2

Modulator of Wnt signaling

PT

0.604

CE

0.602

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POSTN

D13666

GC8557 3

Periostin, osteoblast specific factor

Cell attachment and adhesion

0.599

ZNF385D

NM_0246 97

GC1895 74

Zinc finger protein 385D

Nucleic acid binding

0.596

ATP8B2

AB032963

GC1512 63

ATPase, class I, type 8B, member 2

Nucleotide binding

0.595

C10orf72

AL080114

GC8320 4

Chromosome 10 Protein binding open reading frame 72

0.594

CHI3L1

M80927

GC1792 12

Chitinase 3-like Important role in the 1 (cartilage capacity of cells to glycoprotein-39) respond to and cope with enviromental changes

0.594

CNN1

D17408

GC3720 4

Calponin 1, basic, smooth muscle

Actin binding

0.591

COL1A2

AA628535

GC1492 50

Collagen, type I, 

Protein binding

0.591

LBP

0.591

ODF2

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0.601

GC1829 70

Lipopolysacchar ide binding protein

Affinity enhancer for CD14, facilitating its association with lipopolysaccharides

AL138382

GC1653 90

Outer dense fiber of sperm tails 2

Structural molecule activity

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NM_0041 39

0.591

CLCA2

NM_0065 36

GC1847 52

Chloride channel accessory 2

May act as a tumor suppressor in breast and colorectal cancer.

0.59

PPAPDC3

BC006362

GC1725 54

Phosphatidic acid phosphatase type 2 domain containing 3

Negative regulator of myoblast differentiation.

0.59

DCTN3

W26651

GC3084 0

Dynactin 3 (p22)

Involved in spindle assembly and cytokinesis

0.589

RBP4

NM_0067

GC1849

Retinol binding protein 4,

Retinol transporter

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plasma

activity

BGN

NM_0017 11

GC1811 89

Biglycan

May be involved in collagen fiber assembly

0.588

MOG

U64565

GC2827 5

Myelin oligodendrocyte glycoprotein

Mediates homophilic cell-cell adhesion

0.586

SMAP2

AI702142

GC7136 0

Small ArfGAP2

ARF GTPase activator activity

0.583

PSMB9

AI758695

GC7306 4

Proteasome (prosome, macropain) subunit, beta type, 9 (large multifunctional peptidase 2)

Threonine-type endopeptidase activity

0.58

LDB2

NM_0012 90

GC1808 89

LIM domain binding 2

Transcription factor binding transcription factor activity

0.578

IL17B

NM_0144 43

GC1860 79

Interleukin 17B

Stimulates the release of tumor necrosis factor  and IL-1-.

0.573

EPS15L1

GC1674 31

Epidermal growth factor receptor pathway substrate 15like

Role in receptormediated endocytosis.

GC1747 19

Hypothetical protein LOC284542

Unknown

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44 0.589

ED

AV710549

LOC284542

BF060736

PT

0.573

AC

CE

Reverse COMPAR E (Sensitiv e genes)

26

C1orf86

H41433

GC1301 8

Chromosome 1 open reading frame 86

Polyubiquitin binding

-0.632

ZRANB1

AW26983 6

GC1693 05

Zinc finger, RAN-binding domain containing 1

Ubiquitin thiolesterase activity/positive regulator of Wnt signaling pathway

-0.592

MGC70870

AA278816

GC4284 3

C-terminal binding protein 2 pseudogene

Unknown

-0.59

CTBP2

AF016507

GC5534 0

C-terminal binding protein 2

Transcription corepressor activity

-0.586

CCND1

T89175

GC1186 4

Cyclin D1

Cyclin-dependent protein serine/threonine kinase regulator activity

-0.585

LOC1002876 15

AI700233

GC7120 5

Hypothetical protein LOC100287615

Unknown

-0.574

CMTM4

W46185

GC1574 4

CKLF-like MARVEL transmembrane domain containing 4

Cytokine activity

-0.552

LARP1B

AK027164

GC1636 52

La ribonucleoprote in domain family, member 1B

Nucleic acid binding

ED

M

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-0.635

KCNQ4

H26683

GC1268 3

Potassium voltage-gated channel, KQTlike subfamily, member 4

Delayed rectifier potassium channel activity

-0.534

DOCK6

AI198543

GC6034 9

Dedicator of cytokinesis 6

Guanyl-nucleotide exchange factor activity

-0.53

MYL12B

U26162

GC1911

Myosin, light chain 12B,

Myosin regulatory

CE

PT

-0.539

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regulatory

subunit

SMC5

N70436

GC1509 3

Structural maintenance of chromosomes 5

DNA double-strand repair

-0.524

LOC285147

H23213

GC8711 6

Hypothetical protein LOC285147

Unknown

-0.524

ZNF205

AF060865

GC3793 6

Zinc finger protein 205

Transcriptional regulation

-0.523

SIK2

AA142956

GC4124 1

Salt-inducible kinase 2

Activates insulin signaling pathway

-0.519

TMED5

N49615

GC1432 3

Transmembrane emp24 protein transport domain containing 5

vesicular protein trafficking, maintenance of Golgi apparatus

-0.519

AGAP1

AB029022

GC3768 1

ArfGAP with GTPase domain, ankyrin repeat and PH domain 1

GTPase-activating protein

-0.518

RBMS2

R99202

GC1310 7

RNA binding motif, single stranded interacting protein 2

Nucleotide binding

-0.513

TM9SF3

N93209

GC1531 8

Transmembrane Unknown 9 superfamily member 3

ED

M

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76 -0.526

ST8SIA3

N62107

GC1470 3

ST8 alpha-Nacetylneuraminide 2,8sialyltransferase 3

-Nacetylneuraminate 2,8-sialyltransferase activity

-0.512

HSPC159

AK025603

GC1632 55

Galectin-related protein

Carbohydrate binding

-0.507

SPATA20

H15544

GC1129 3

Spermatogenesi s associated 20

Fertility regulation

-0.506

ZNF652

AA057773

GC1725

Zinc finger

Transcriptional

CE

PT

-0.512

AC

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protein 652

repressor

MRPL17

AI141700

GC5952 6

Mitochondrial ribosomal protein L17

Structural constituent of ribosome

-0.504

C13orf1

N30354

GC1402 4

Chromosome 13 Protein binding open reading frame 1

-0.501

TAC1

N47310

-0.5

GCOM1

N51713

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1 0.506

GC1448 5

Tachykinin, precursor 1

Active peptides which excite neurons

GC1440 0

GRINL1A complex locus RNA

Unknown

Only correlations with cut-offs of R > 0.5 and R < −0.5 were considered. Gene function information was retrieved from Gene Card database (http://www.genecards.org)

Partition ≤-4.80 M >-4.80 M P=0.00437 (χ2test)

Cluster 1 3 8

Cluster 2 5 11

Cluster 3 8 2

Cluster 4 5 0

AC

CE

PT

ED

Sensitive Resistant

M

Table 3. Separation of clusters of cancer cell lines obtained by hierarchical cluster analysis for reserpine. The log10 IC50 median value (M) of reserpine was used as cutoff.

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A. Drug Class Profile

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Antihormones Alkylating Drugs mTOR Inhibitors DNA Topoisomerase II Inhibitors (Anthracyclines,… Antimetabolites Tyrosin Kinase Inhibitors DNA Topoisomerase I Inhibitors (Camptothecins) Platinum Compounds Tubulin Inhibitors Epigenetic Inhibitors

0

10

20

30

40

50

B. Antihormones Anastrozol 0.5 0.4 0.3 0.2 0.1 0 -0.1 -0.2

Toremifen

Tamoxifen

Exemestane

Fulvestrant

M

Raloxifene

Megostrol

C. Alkylating Drugs

Azathioprine 0.50000 Thiotepa 0.40000

ED

Streptozocin

Semustine (MeCNU)

Bendamustine

0.30000

Busulfan

0.20000 0.10000

Carmustine (BCNU)

0.00000

Melphalan

AC

CE

PT

Mafosfamide Lomustine (CCNU)

Chlorambucil Dacarbazine Ifosfamide

32

60

AC PT

CE M

Ovarian cancer

-5.2

Leukemia

Brain tumor

-4.2

-4.4

CR IP T

AN US

-5

Renal cancer

-4.8

Melanoma

log10IC50 (M) -4.6

Lung cancer

Colon Cancer

ED

ACCEPTED MANUSCRIPT

33

CR IP T

Reserpine

1

1

2

2

3

3

AN US

Cell line: SR RPMI-8226 K-562 MOLT-4 CCRF-CEM

Tumor type: Melanoma Melanoma Melanoma Melanoma CNS tumor CNS tumor CNS tumor CNS tumor Lung cancer Leukemia

Cell line: KM12 SW-620 HT29 OVCAR-4 OVCAR3 NCI-H460 OVCAR-5 HCT-15 HCC-2998 NCI-H322M

Cell line: UACC-62 UACC-257 SK-MEL-5 M14 SF-539 SF-295 U251 SNB-19 HOP-92 HL-60 (TB)

M

Tumor type: Leukemia Leukemia Leukemia Leukemia Leukemia

3

ED

Cell line: MALME-3M NCI-H23 HOP-62 ACHN OVCAR-8 CAKI-8 786-0 IGROV1 TK-10 UO-31 SK-MEL-2 HCT-116 NCI-H522 NCI-H226 A549/ATCC

Tumor type: Colon cancer Colon cancer Colon cancer Ovarian cancer Ovarian cancer Lung cancer Ovarian cancer Colon cancer Colon cancer Lung cancer

PT

Tumor type: Melanoma Lung cancer Lung cancer Renal cancer Ovarian cancer Renal cancer Renal cancer Ovarian cancer Renal cancer Renal cancer Melanoma Colon cancer Lung cancer Lung cancer Lung cancer

CE

4

1

AC

2

ACCEPTED MANUSCRIPT

4

4