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The biochemical and proteomic analysis of a potential anticancer agent: Palladium(II) Saccharinate Complex of Terpyridine acting through double strand break formation Zelal Adiguzel, Ahmet Tarik Baykal, Omer Kacar, Veysel T. Yilmaz, Engin Ulukaya, and Ceyda ACILAN J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/pr5006718 • Publication Date (Web): 27 Aug 2014 Downloaded from http://pubs.acs.org on August 28, 2014

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Journal of Proteome Research

The biochemical and proteomic analysis of a potential anticancer agent: Palladium(II) Saccharinate Complex of Terpyridine acting through double strand break formation

Zelal Adiguzel1‡, Ahmet Tarik Baykal2‡, Omer Kacar1, Veysel T. Yilmaz3, Engin Ulukaya4 and Ceyda Acilan1*

1

TUBITAK, Marmara Research Center, Genetic Engineering and Biotechnology

Institute, Gebze/Kocaeli, 41470, TURKEY 2

Medipol University, Medical School, Kavacık/Istanbul, 34810, TURKEY

3

Uludag University, Faculty of Arts and Sciences, Department of Chemistry, 16120,

Bursa, TURKEY 4

Uludag University, Medical School, Department of Medical Biochemistry, 16120,

Bursa, TURKEY ‡

These authors contributed equally to this work.

*To whom correspondence should be addressed: Dr. Ceyda Acilan E-mail: [email protected] Tel: +90 262 677 3354 Fax: +90 262 641 2309 1 ACS Paragon Plus Environment


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ABSTRACT: Metal-based chemotherapeutic drugs are widely used as an effective method to defeat various cancers. In this study, the mechanism of action of a novel therapeutic agent, [Pd(sac)(terpy)](sac)•4H2O (sac = saccharinate, and terpy = 2,2':6',2''proteomic changes in response to this agent, we performed a nano LC-MS/MS analyses in human breast cancer cells (MDA-MB-231). 30 proteins were identified to be differentially expressed more than 40% after drug treatment. Many cellular pathways were affected including proteins involved in DNA repair, apoptosis, energy metabolism, protein folding, cytoskeleton, pre-mRNA maturation or protein translation. The changes in protein expression were further verified for XRCC5, which plays a role in double strand break (DSB) repair; and Ubiquitin, which is involved in protein degradation and apoptosis. The elevated XRCC5 levels were suggestive of increased DSBs. The presence of DSBs was confirmed by smearing of plasmid DNA in vitro and induction of γH2AX foci in vivo. There was also increased intracellular reactive oxygen species (ROS) formation, as detected by 2’,7’ – dichlorofluorescein diacetate (DCFDA) staining. Scavenging ROS by N-Acetylcysteine rescued cell death in response to Pd(II) treatment, potentially explaining how the Pd(II) complex damaged the DNA. The details of this analysis and the significance will be discussed during the scope of this work.

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INTRODUCTION: The Platinum (Pt) based anticancer drug, cisplatin, has gained wide clinical application in various types of cancers including breast, ovarian, testicular, prostate, small cell lung, germ cell cancers, neuroblastoma, sarcoma or lymphoma (1, 2). Despite its broad efficacy and success, its application has been complicated due to its

side

effects

such

as

toxicity

(nephrotoxicity,

neurotoxicity,

ototoxicity,

myelotoxicity, peripheral neuropathy, hematological toxicity and emetogenity) as well as intrinsic and acquired resistance of cancer cells to cisplatin (3, 4). That is why, ever since its discovery, major efforts have been devoted to synthesize alternative molecules with better pharmacological properties (5). Palladium (Pd) based compounds have gained the interest of many researchers as they exhibit similar coordination chemistry and geometry compared to Platinum containing drugs (6). Several studies reported cytostatic and cytotoxic effects of Pd(II) complexes to at least comparable levels to cisplatin (7-12). However, unlike cisplatin, Pd(II) compounds can be expected to exhibit less nephrotoxicity (13). Furthermore, better solubility of Pd(II) complexes appear to make them more attractive as anticancer agents (14). Recently, we have reported a promising Palladium based compound that seemed to be more potent than cisplatin in breast cancer cells in vitro and in Balb/c mice in vivo (9, 15). This compound also effectively disrupted tubule formation of MDA-MB-231 cells on matrigel suggesting a putative anti-invasive activity (9). Thereby, this molecule warranted further evaluation for its use in vivo. The mechanism of action for this novel Pd(II) complex is not yet fully understood. There is evidence that it induces cell death mainly through apoptosis involving DR4



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and DR5 gene expression (9), yet no other molecule has been identified. In order to elucidate the biochemical mechanisms responsible for the anticancer activity of this compound, we have performed a nano LC-MS/MS analysis and investigated the global proteomic changes in response to Pd(II) treatment. Many pathways were found to be affected including proteins having roles in DNA repair, apoptosis, energy metabolism, protein folding, cytoskeleton, pre-mRNA maturation or protein translation. The expression changes in a subset of these proteins were verified by western blotting (Ubiquitin and XRCC5/Ku80). The upregulation in the level of XRRC5 protein, which is involved in DNA double strand breaks (DSB) repair through joining non-homologous DNA ends (16), was suggestive of DNA DSB induction in response to Pd(II) complex. Indeed, DSBs were formed both in vitro and in vivo as determined by plasmid incubation assay and by examining the presence of γH2AX foci. We also found that drug treatment lead to an increase in reactive oxygen species (ROS) formation. Addition of antioxidants to scavenge ROS rescued cell viability almost completely, indicating that the new Pd(II) complex induced its toxic effect through ROS intermediates.

Keywords: Palladium(II) Saccharinate Complex with Terpyridine, anticancer drugs, DNA double strand breaks, reactive oxygen species, non-homologous end joining, apoptosis, proteomics


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MATERIALS AND METHODS: Cell Culturing: MDA-MB-231, A549 and Hela cells were cultured as monolayers in Dulbecco’s Modified Eagle Medium-F12 (DMEM/F12, Sigma-Aldrich, #D0547) supplemented with 5% FBS (Biochrom, #S0415), and penicillin (100 units/ml)-streptomycin (100 µg/ml) (Biochrom, #A2212) at 37ºC in 5% CO2. Cells were harvested using Trypsin (0.05%, Biochrom, L2143). Treatment of Cells with the Pd(II) Complex: The

synthesis,

characterization

and

chemical

properties

of

[Pd(sac)(terpy)](sac)•4H2O (sac = saccharinate, and terpy = 2,2':6',2''-terpyridine) was described elsewhere (15). MDA-MB-231 cells were seeded on 100 mm and 96well plates in parallel the day before the experiment. Cells on 100 mm plates were treated with 6.25 µM Pd(II) complex for 48 h. The cells on 96-well plates were used for determination of cell viability after drug treatment. Determination of Cell Viability: 8 × 103 cells were seeded into 96 well plates. Following overnight culture at 37ºC, 5% CO2, cells were treated with freshly prepared Pd(II) complex at concentrations of 6.25, 12.5, 25, 50 µM in culture medium for indicated periods and the viability was measured using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue) assay (Sigma, Cat # M 5655). Briefly, 10 µl of MTT solution (5 mg/ml) was added to each well containing 90 µl of medium. Following 4 h incubation at 37ºC, 50 µl solubilizing buffer (10% sodium dodecyl sulfate dissolved in 0.01 N HCl) was added and samples were measured at 570 nm. The cell survival ratio was expressed as a percentage of the untreated control cells.



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N-Acetylcyteine (NAC) Treatment 8 × 103 cells were seeded into 96 well plates, and incubated overnight at 37ºC, 5% CO2. NAC (Sigma, cat # A9165, 1 mM) was freshly prepared in culture medium and cells were pre-incubated in NAC for 24 h. Pd(II) complex was also prepared fresh at indicated concentrations in medium containing 1 mM NAC. Following 24 h drug treatment, cell viability (MTT assay) and total ROS (DCFDA Assay) were measured. The differences were expressed as percentage of untreated samples. DNA Fragmentation Assay: 1 × 106 cells were seeded on 100 mm petri dishes. Following overnight culture at 37°C, 5% CO2, cells were treated with freshly prepared Pd(II) complex (25 µM) dissolved in growth medium for 24, 48 and 72 h. Both the floating cells and the attached cells were collected and centrifuged (1000 g, 5 min, room temperature (RT)). The pellet was washed with cold PBS and dissolved in 120 µl lysis buffer (10 mM Tris pH:7.4, 100 mM NaCl, 25 mM EDTA, 1% N-lauryl sarcosine) with gentle vortexing. 4 µl of proteinase K (10 µg/µl) was added to each tube and incubated at 45°C for 2 h. Following addition of RNAse A (2 µl, 10 µg/ml), the lysates were further incubated for 1 h at RT. The DNA was slowly run on 2% agarose gels stained with SYBR green for 4 h at 60 V. Immunofluorescence Staining: 2 × 104 cells were seeded on glass coverslips and treated with freshly prepared Pd(II) complex following overnight culture at 37ºC, 5% CO2. Cells were stained as described

in

(17).

Briefly,

for

γH2AX

staining;

cells

were

fixed

in

4%

paraformaldehyde/PBS (15 min, RT), permeabilized with 0.3% Triton X-100/PBS (15 min, on ice), blocked in 0.2% gelatin/PBS (1 h, RT), stained for γH2AX (Cell Signaling, #9718S, 1:400 dilution, at 4ºC, overnight), and labeled with FITC 6 ACS Paragon Plus Environment

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conjugated secondary antibodies (Abcam, #97068, 1:250 dilution, 1 h, RT).

For

Ku80 staining; cells were fixed in -20ºC methanol (5 min), permeabilized with 0.2% Triton X-100/PBS (5 min, RT), blocked with 1% BSA/PBS (30 min, RT), stained for Ku80 (BD Biosciences, USA #611360, 1:400 dilution, at 4ºC, overnight) and labeled with FITC conjugated secondary antibodies (Abcam, # ab60316, 1:250 dilution, 1 h, RT). Coverslips were reverted on mounting medium containing Hoescht dye and visualized using a fluorescence microscope (Leica, DMI 6000). Immunoblotting: Total cell lysates (20 µg) were loaded into 10% SDS-PAGE gels and run for 1.5 h at 150 V. Proteins were transferred to a PVDF membrane, blocked with 3% BSA dissolved in PBS for 1 h at RT, incubated with γH2AX (Cell Signaling, #9718S, 1:400 dilution), Ubiquitin (Abcam, # ab19247, 1:1000 dilution), β-Tubulin (Abcam, # ab6046, 1:500 dilution), or β-actin (Abcam, # ab8227, 1:2000 dilution,) antibodies (all at 4ºC, overnight) and secondary antibodies (Pierce, #32430ab, 1 h, RT). Proteins were visualized by enhanced chemilluminescence (Thermo Scientific, Cat # 34080). In Vitro Analysis of Pd(II)/DNA Interaction: 120 ng of plasmid DNA was incubated with either Pd(II) complex or cisplatin at indicated final concentrations for 20 h, at RT. Reactions were set in a total 20 µl. Sodium azide (0.02%, Sigma, #S-2002) was added to the tubes with the two highest drug concentrations (50 and 100 µM) at a 0.005% final concentration. The DNA was run on 1% SYBR Green stained agarose gels. Detection of Total ROS via 2’,7’ – dichlorofluorescein diacetate (DCFDA): 8 × 103 cells were seeded into 96 well plates. Following overnight culture at 37ºC, 5% CO2, cells were treated with freshly prepared Pd(II) complex at indicated doses and durations. Cells were washed once in PBS, and incubated in culture medium



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containing 5 µM DCFDA (Sigma, cat # D6883) for 20 min at 37ºC. Plates were read at 485 nm (excitation), 535 nm (emission). The fluorescence intensity was corrected for viable cells left in each well, as a fraction of cells were lost after Pd(II) treatment. Sample Preparation for Analysis: Cells were grown on 100 mm plates until 80% confluency and either were left untreated or treated with 6.25 µM of Pd(II) complex. Four independent plates of untreated cells and three independent biological repeats of treated cells were prepared for this analysis. A modified protocol from (18) was used for sample preparation. Briefly, cells were lysed with UPX extraction buffer (Expedeon) following two washes with ice cold 50 mM ammonium bicarbonate solution. Trypsinization was performed as described in the Filter Aided Sample Preparation Protocol (FASP) (19). Briefly, protein extract (50 µg) was washed with urea (6 M) in a 30 kDa cut-off spin column and alkylated with iodoacetamide (IAA, 10 mM) in the dark (20 min, RT). IAA was removed from the samples by washing with 6 M urea and the urea was removed with ammonium bicarbonate solution. The samples were trypsinized overnight (1:100 trypsin to protein ratio). Peptides were eluted from the spin column and protein concentration was measured using a nanodrop spectrometer and adjusted to a concentration of 100 ng/µl. LC-MS/MS Analysis and Database Search: Both the LC-MS/MS analysis and identification of proteins were performed following previous protocols published by our laboratory (20). Briefly, tryptic peptides (500 ng in 5 µl for each experimental condition) were analyzed by the LC-MS/MS system (nanoACQUITY ultra pressure liquid chromatography (UPLC) and SYNAPT G1 high definition mass spectrometer with nanolockspray ion source (Waters)). Equilibration of the columns was done using a 97% mobile phase A (0.1% formic acid in LC-MS


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grade water, Merck) at a column temperature of 45°C. Peptides were separated from the trap column (Symmetry C18, 5 µm, 180 µm i.d. x 20 mm, Waters) by gradient elution onto an analytical column (BEH C18, 1.7 µm, 75 µm i.d. x 250 mm, Waters) at 300 nl/min flow rate with a linear gradient from 5-40% mobile phase B (0.1 formic acid in hypergrade acetonitrile, Merck) over 90 min. All samples were analyzed in triplicate to eliminate technical errors. Protein Identification Criteria: The MS and MS/MS functions were applied over 1.5 sec intervals with 6 V low energy and 15-40 V high energy collusions. Data independent acquisition mode (MSE) was performed by operating the instrument at positive ion V mode. Glufibrinopeptide (internal mass calibrant) was infused every 45 sec at 300 nl/min flow rate. The m/z values over 50-1600 were analyzed. ProteinLynx Global Server v 2.5 (Waters) was used for tandem mass data extraction, charge state deconvolution and deisotoping. For protein identification, homo sapiens protein database from Uniprot (June1st 2012, 25899 entries) was used with the IDENTITYE algorithm. The Apex3D data preparation parameters were set to 0.2 min chromatographic peak width, 10.000 MS TOF resolution, 150 counts for low energy threshold, 50 counts for elevated energy threshold, and 1200 counts for the intensity threshold. Databank search query was set to minimum 3 fragment ion matches per peptide, minimum 7 fragment ion matches per protein, minimum 1 peptide matches per protein and 1 missed cleavage. Carbamidomethyl-cysteine fixed modification and acetyl N-TERM, deamidation of asparagine and glutamine, and oxidation of methionine variable modifications were set. Scaffold (version Scaffold_3.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Identification of peptides was accepted if they could be established at greater than 95.0% probability 9 ACS Paragon Plus Environment


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as specified by the Peptide Prophet algorithm (21). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm (22). Only the protein IDs that satisfy the above criteria were listed and the corresponding information regarding peptide sequences and protein identification probabilities were provided in the supplementary file (Supplemental Table 1). The false positive rate for the identified proteins was calculated to be 0.7% based on protein prophet algorithm in Scaffold. All the listed proteins were identified by at least 2 unique peptide sequences that were tabulated in the supplementary file (Supplemental Table 1). Quantification of the protein expression changes was done with Progenesis LC-MS software V4.0 (Nonlinear Dynamics). Normalization across samples was based on total ion intensity. Similar proteins were grouped and quantitative value is given for the one with the highest score. Protein quantitation was done with only the nonconflicting peptide features.


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RESULTS: Figure 1 illustrates the molecular structure of the Pd(II) complex used in this study (Molecular weight: 749.08 g/mol). The synthesis, characterization and the X-Ray structure of this complex were described previously (15). The complex was also shown to be toxic to breast cancer cells and to induce cell death through apoptosis (9), which was further verified in this study in MDA-MB-231 breast cancer cell line using formerly determined time points (Supplemental Figure 1). In order to evaluate which molecular pathways were differentially regulated in response to drug treatment, cells were subjected to 6.25 µM Pd(II) complex for 48 h. The optimum effective dose and incubation time was chosen based on cell viability, where the cells were affected, but were mostly alive. Protein extracts were prepared from treated and untreated cells, tyrpsinized, and loaded on to a nanoACQUITY UPLC system (Waters Corp.) coupled to SYNAPT High definition mass spectrometer (Waters Corp.). The data were collected from three independent biological repeats and three injections of each repeat. 681 proteins in 356 protein groups were qualitatively identified and quantification data for 335 protein groups was acquired. Normalization of the peptide intensities was based on total protein amount loaded in the column. 163 proteins out of 335, were found to be significantly differentially regulated using Progenesis LC-MS. Furthermore, intensity cut-off was set to 40% and only the proteins that are up or down regulated more than the 40% cut-off is reported. Following the limiting criteria above, we identified 30 differentially regulated proteins that were statistically significant (p-value 25 µM) even at 24 h ((9) and data not shown). However, in the case of lung cancer, the same Pd(II) compound resulted in significant cytotoxicity at even much lower doses (2. Proteins in bold were identified to be significantly differentially regulated in this study and others were added by IPA for network analysis. The table shows the focus molecules in each network, top functions based on these focus molecules, the score and the number of focus molecules in the network. Networks 1 and 2 are drawn in Figure 2 and Supplemental Figure 2.


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Table 1. Differentially regulated proteins identified in this study with a 40% cutoff for expression change Accession Number

Fold Regulation p-Value Description Change

Regulation of Apoptosis -07

P55060

7.27

↓

1.07×10

XPO2 Exportin 2

P09382

1.65

↓

6.21×10-04 LEG1 Galectin 1

P62158

2.35

↓

9.70×10-06 CALM Calmodulin

O76021

1.83

↓

1.06×10

P09104

3.17

↓

8.39×10-12 ENOG Gamma enolase

Q13200

1.59

↑

1.41×10

P0CG48

4.72

↑

6.27×10-06 UBC Polyubiquitin C

P13010

1.47

↑

5.43×10

P09429

1.40

↓

3.55×10-07 HMGB1 High mobility group protein B1

Q08945

1.42

↑

1.42×10-07 SSRP1 FACT complex subunit SSRP1

Q9BQE3

1.64

↓

3.33×10

Q6PEY2

1.57

↑

2.52×10-14 TBA3C Tubulin alpha 3C D chain

P29966

1.41

↓

1.80×10

P09651

2.69

↓

1.69×10

P09651-2

2.61

↓

3.01×10-04 ROA1 Isoform A1 Heterogeneous nuclear ribonucleoprotein A1

2.13

↓

2.14×10-09 1A02 HLA class I histocompatibility antigen A 2 alpha chain

Proliferation -04

RL1D1 Ribosomal L1

Protein Degradation -06

PSMD2 26S proteasome non ATPase regulatory subunit 2

DNA Repair -10

XRCC5 X ray repair cross complementing protein 5

Cytoskeleton -02

TBA1C Tubulin alpha 1C chain

-02

MARCS Myristoylated alanine rich C kinase substrate

-04

ROA1 Heterogeneous nuclear ribonucleoprotein A1

mRNA and Protein Maturation

HLA proteins P01892


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2.00

↓

2.33×10-02 1C07 HLA class I histocompatibility antigen Cw 7 alpha chain

P53618

1.91

↑

2.71×10-06 COPB Coatomer subunit beta

Q99829

1.74

↑

1.47×10-03 CPNE1 Copine 1

P18669

1.44

↑

2.05×10

P52789

1.79

↑

1.98×10-06 HXK2 Hexokinase 2

P68431

2.09

↓

1.19×10

Q71DI3

1.84

↓

5.96×10

P62805

1.42

↓

5.80×10-03 H4 Histone H4

Q05639

2.76

↑

1.77×10

P60842

1.47

↑

2.62×10

P05386

1.62

↓

1.02×10-04 RLA1 60S acidic ribosomal protein P1

P98179

1.68

↓

1.53×10

O75531

1.51

↓

2.16×10

P51571

1.51

↓

5.80×10-03 SSRD Translocon associated protein subunit delta

P10321 Membrane Traficking

Energy Metabolism -04

PGAM1 Phosphoglycerate mutase 1

Histones -05

H31 Histone H3 1

-05

H32 Histone H3 2

Protein Translation -03

EF1A2 Elongation factor 1 alpha 2

-06

IF4A1 Eukaryotic initiation factor 4A I

Others -07

RBM3 Putative RNA binding protein 3

-05

BAF Barrier to autointegration factor



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Figure 1. The molecular structure for [Pd(sac)(terpy)](sac)•4H2O (sac = saccharinate, and terpy = 2,2':6',2''-terpyridine) used in this study 459x305mm (96 x 96 DPI)

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Figure 2. Most high-scored network (Network1): Pathway generated by ingenuity pathway analysis with proteins identified as specific target antigens. (A) Color code indicates fold change; Green: downregulation, Red: upregulation. Darker colors indicate higher fold changes. Colorless proteins were not detected in our analysis but added to the network by the IPA algorithm. Proteins are placed based on their cellular location; labeled extracellular space, cytoplasm, and nucleus. 23 out of 30 proteins were included in this network with a score of 69. (B) IPA legends for path designer shapes and edge types. 584x762mm (96 x 96 DPI)



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Figure 2. Most high-scored network (Network1): Pathway generated by ingenuity pathway analysis with proteins identified as specific target antigens. (A) Color code indicates fold change; Green: downregulation, Red: upregulation. Darker colors indicate higher fold changes. Colorless proteins were not detected in our analysis but added to the network by the IPA algorithm. Proteins are placed based on their cellular location; labeled extracellular space, cytoplasm, and nucleus. 23 out of 30 proteins were included in this network with a score of 69. (B) IPA legends for path designer shapes and edge types. 372x322mm (96 x 96 DPI)


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Figure 3. Confirmation of differential protein expression For verification of Ubiquitin upregulation (A), cells were either untreated (lanes 1-3, labelled control samples) or treated with 6.25 µM Pd(II) complex for 48 h (lanes 4-6, labelled Pd(II) treated samples), lysed and immunoblotted. Results for three independent experiments are shown. α-actin was used as loading control. For verification of XRCC5 upregulation (B), cells were treated with 25 µM Pd(II) complex for 48 h and stained. Same camera settings were used to take all images. DNA was visualized via Hoechst 33342 staining. 409x631mm (96 x 96 DPI)



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Figure 4. In vitro Analysis of Pd(II) complex/DNA Interactions: Plasmid DNA was incubated with different concentrations of either cisplatin (top panel) or with Pd(II) compound (bottom panel), (0 to100 µM as indicated on the lanes) for 20 h at room temperature in ddH2O. DNA was visualized on SYBR green stained 1% agarose gel. Uncut plasmid DNA was used as control and loaded twice (first and last lanes). “0 µM” treatment lane indicates diluted plasmid DNA incubated at RT for 20 h without any treatment. Different forms of plasmid DNA are indicated on the left of the image. Cisplatin treatment did not result in any smearing, but rather lead to faster migration indicating further supercoiling (top panel). On the other hand, DNA smearing was observed starting at 12.5 µM of Pd(II) treatment, suggesting that Pd(II) complex can induce DNA DSBs. The smearing appeared at lower molecular weight as the concentration of Pd(II) increased and completely disappeared by 100 µM, probably due to further degradation of DNA. Addition of NaN3 rescued smearing suggesting ROS intermediates in during Pd(II) induced DNA damage. 582x523mm (96 x 96 DPI)


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Figure 5. DNA DSB induction following Pd(II) treatment in cell culture Following 24 h treatment with the Pd(II) complex (25 or 50 µM) at 37oC, cells were either fixed in 4% paraformaldehyde (A) or lysed (B) and stained (A) or blotted (B) for γH2AX to detect DSBs. Untreated cells exhibited no foci formation (0 µM). Foci formation was evident indicated by arrows in treated samples confirming that Pd(II) can induce DNA DSBs (A). Blue color indicates DNA with Hoechst 33342 stain, and green indicates γH2AX staining. There was also a marked increase in γH2AX in treated samples detected via western blotting (B). β-Tubulin was used as loading control. 578x472mm (96 x 96 DPI)



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Figure 6. Rescue of ROS formation and cell viability upon NAC treatment Cells were either untreated or preincubated with NAC for 24 h before the addition of Pd(II) complex at indicated concentrations (x-axis). Total ROS levels (y-axis) were increased upon drug addition in control samples (grey bars) as measured using DCFDA, which was reduced to background levels in samples where NAC was added (black bars) (A). Presence of NAC also rescued cell viability (y-axis) (B), suggesting that ROS plays a role in Pd(II) induced cytotoxicity. 309x525mm (96 x 96 DPI)


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Figure 7. Proposed model for the cytotoxic action of Pd(II) complex under study Our data suggest that Pd(II) treatment lead to ROS that damage the DNA causing DSBs. Cells respond to this damage by increase in repair proteins, as determined by the elevation in XRCC5 expression, an NHEJ protein. Insufficient repair in cases of higher doses or longer exposure times of the drug lead to apoptosis that involve ubiquitination of several proteins. 324x489mm (96 x 96 DPI)


Palladium(II) Saccharinate Complex of Terpyridine ... - ACS Publications

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