Cancer Biology & Therapy Riproximin's activity ...

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Jun 11, 2014 - promised, as exemplified by the increased expression of Bcl-2 and .... Figure 1. cytotoxic activity of riproximin (Rpx) in pancreatic ductal ade-.
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Riproximin’s activity depends on gene expression and sensitizes PDAC cells to TRAIL a

a

a

a

Hassan Adwan , Ahmed Murtaja , Khamael Kadhim Al-Taee , Asim Pervaiz , Thomas b

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Hielscher & Martin R Berger a

Toxicology and Chemotherapy Unit; German Cancer Research Center (DKFZ); Heidelberg, Germany b

Division of Biostatistics; Statistics for Translational Oncology; DKFZ; Heidelberg, Germany Published online: 11 Jun 2014.

Click for updates To cite this article: Hassan Adwan, Ahmed Murtaja, Khamael Kadhim Al-Taee, Asim Pervaiz, Thomas Hielscher & Martin R Berger (2014) Riproximin’s activity depends on gene expression and sensitizes PDAC cells to TRAIL, Cancer Biology & Therapy, 15:9, 1185-1197, DOI: 10.4161/cbt.29503 To link to this article: http://dx.doi.org/10.4161/cbt.29503

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Research Paper

Research Paper

Cancer Biology & Therapy 15:9, 1185–1197; September 2014; © 2014 Landes Bioscience

Riproximin’s activity depends on gene expression and sensitizes PDAC cells to TRAIL Hassan Adwan1, Ahmed Murtaja1, Khamael Kadhim Al-Taee1, Asim Pervaiz1, Thomas Hielscher2, and Martin R Berger1,* Toxicology and Chemotherapy Unit; German Cancer Research Center (DKFZ); Heidelberg, Germany; 2Division of Biostatistics; Statistics for Translational Oncology; DKFZ; Heidelberg, Germany

1

Riproximin (Rpx) is a type II ribosome inactivating protein, which was investigated for its activity in pancreatic ductal adenocarcinoma (PDAC) in a panel of 17 human and rat PDAC cell lines and in rat pancreatic cancer liver metastasis. Cytotoxicity in response to Rpx was determined by MTT assay, apoptosis by flow cytometry and qRT-PCR for apoptosis related genes, and the modulation of the transcriptome was monitored by micro array analysis. The combination effect of Rpx and TRAIL was assessed by MTT assay. Rpx showed high but varying cytotoxicity in PDAC cells. Based on overall gene expression, the sensitivity of these cells was linked to genes involved in apoptosis. Furthermore, based on the affinity of Rpx for CEA, the expression of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) genes was significantly related to Rpx’s cytotoxicity in cells with CEACAM gene expression. Exposure of Suit2–007 cells to Rpx induced the mRNA expression of members of signaling pathways initiating from most death receptors, and down modulation of TRAIL. Apoptosis was increased as shown by FACS analysis. Combination of Rpx with TRAIL resulted in a synergistic cytotoxic effect in human Suit2–007 and rat ASML cells, as evidenced by a 6-fold lower tumor cell survival than expected from an additive combination effect. Treatment of BDX rats bearing intra-portally implanted Suit2–007 cells showed a highly significant anticancer effect and indicated an application of Rpx against pancreatic cancer metastasis to the liver. These data favor further evaluation of Rpx as anticancer agent in PDAC.

Introduction Riproximin (Rpx) is a recently discovered plant lectin, which belongs to the group of cytotoxic type II ribosome inactivating proteins (RIP).1 These proteins consist of two chains, which exhibit complementary properties.2 The A chain is an rRNA N-glycosidase, which causes depurination of the 28S rRNA in a catalytic manner and thus leads to transcriptional arrest by inhibiting protein synthesis.3 The B-chain binds to glycan structures and is prerequisite for internalization of the RIP type II molecule.3 Accordingly, the presence of certain carbohydrates on the cell surface determines their affinity to a given cell. The well-known type II RIP ricin, e.g., reacts with galactose residues,3 but the type II RIP mistletoe lectin I is more specific and reacts preferentially with terminally sialylated neolacto series gangliosides.4 For riproximin, a significant affinity was found for bi-and tri-antennary N-glycan structures including CEA, as well as to O-glycan structures with three consecutive O-linked GalNAcα moieties.5 The high toxicity after internalization of these RIPs is related to cell death resulting from apoptosis induction.6-8 Pancreatic ductal adenocarcinoma (PDAC) has one of the worst survival rates of all malignancies. Despite continued efforts over the past decades to improve diagnosis and treatment, the prognosis of patients with PDAC is still poor, and incidence rates are virtually identical to mortality rates.9,10 Drug resistance is

thought to be a major reason for the limited benefit of pharmacological therapies. A variety of drug resistance mechanisms has been found in PDAC, including changes in individual genes or signaling pathways, the influence of the tumor microenvironment, and the presence of highly resistant stem cells.11 Among the signaling pathways in PDAC, apoptotic cascades can be compromised, as exemplified by the increased expression of Bcl-2 and Bcl-xL.12 Other examples of mutations leading to altered gene function include mutations of the p53 tumor suppressor gene, of K-ras,13 of the EGF-receptor,14 and many more. Current treatment of PDAC is based on surgery, which is possible in only 20% of cases. In unresectable cases as well as in surgically treated patients who show recurrence, the liver is very often involved, and these patients are left to antineoplastic chemotherapy with or without irradiation. The most effective single drug against advanced PDAC is gemcitabine (GEM), which has modestly improved the median overall survival of PDAC patients. Combination therapy with FOLFIRINOX has enhanced the median overall survival, when compared with GEM.15 The efficacy of chemo-radiation in advanced PDAC is superior to best supportive care and to radiotherapy alone, but more toxic and equally effective in comparison to chemotherapy alone.16 Based on this background, new drugs are needed for the treatment of PDAC. We reasoned that it would be worth investigating

*Correspondence to: Martin R Berger; Email: [email protected] Submitted: 05/28/2014; Accepted: 06/05/2014; Published Online: 06/11/2014 http://dx.doi.org/10.4161/cbt.29503 www.landesbioscience.com Cancer Biology & Therapy 1185

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Keywords: riproximin, type II ribosome inactivating protein, pancreatic ductal adenocarcinoma, TRAIL, synergistic cytotoxicity

the activity of Rpx in PDAC models, because a colorectal cancer liver metastasis model had shown explicit antineoplastic activity.1 Moreover, the affinity of Rpx for carcinoembryonic antigen (CEA)5 suggested that neoplasias expressing this glycoprotein might be preferential targets. Therefore, we determined the sensitivity of 17 PDAC cell lines to Rpx and investigated in a newly established PDAC liver metastasis model, whether Rpx has antineoplastic efficacy against PDAC cells growing in rat liver. The differential sensitivity to Rpx observed in our panel of cell lines caused us to relate the expression profile of these cells with their sensitivity to Rpx. The lectin’s potential to induce apoptosis8 was then basis to monitor the expression of apoptosis related genes in PDAC cells following Rpx treatment. The modulation of gene expression observed in this experiment prompted the choice of TRAIL as combination partner of Rpx, and this combination showed synergistic antiproliferative activity. Here we report our results on the anti-proliferative activity of Rpx in a panel of PDAC cell lines, the antineoplastic effect of Rpx in a new PDAC liver metastasis model, the influence of the genetic profile on the PDAC cells’ sensitivity to Rpx, and the synergistic combination effect of Rpx and TRAIL.

Results Sensitivity of PDAC cell lines to riproximin The antiproliferative efficacy of Rpx was investigated in a panel of 15 human and two rat pancreatic cancer cell lines, which were exposed to Rpx for different periods and concentrations. An overview of the resulting cell growth inhibition is given in Figure 1. The IC50 values ranged from 0.8 pM (MIA PaCa cells) to 60 pM (Colo 357 cells) by a factor of 73. The mean IC50 value derived from all cell lines was 15.1 ± 16.6 pM after 48 h. With increasing time of drug exposure, the IC50 remained

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Figure 1. Cytotoxic activity of riproximin (Rpx) in pancreatic ductal adenocarcinoma (PDAC) cell lines. The cytotoxic efficacy of Rpx was measured after 48 h in a panel of 15 human and two rat PDAC cell lines The IC50 values are given as ratio based on the average of all IC50 values (15.1 ± 16.6 pM), which was set to 1.

about constant in four cell lines (TM34, Patu, MiaPaca, ASML), decreased slightly in three cell lines (AS, Colo, C8.18) and declined significantly in the remaining ten cell lines. Migration studies showed that no specific anti-migratory effect was observed after correcting for proliferation (data not shown). ASML liver metastasis model in BDX rats ASML cells are derived from a pancreatic adeno-carcinoma growing in BDX rats. For mimicking PDAC liver metastasis, ASML cells, which had been transfected with the reporter genes eGFP and luciferase17 were implanted intra-portally to BDX rats. One week after tumor cell transplantation, all rats showed clearly visible tumor growth. At three weeks after tumor cell inoculation, the liver of control rats was distinctly enlarged, corresponding to a clear bioluminescence signal (Fig. 2A). Administration of Rpx to tumor bearing rats started one week after tumor cell implantation and reduced the tumor growth significantly (P ≤ 0.001). At the end of the experimental period, a dose dependent decrease in tumor weight was obtained (53% and 57% following administration of 100 ng and 150 ng riproximin, respectively; see Fig. 2). Gene expression and sensitivity of PDAC cells The mRNA expression profiles of 14 human and one rat PDAC cell lines were analyzed and their mRNA expression levels were correlated in a genome wide analysis with the cell lines’ IC50 values, as indicator of their sensitivity to Rpx. From this association, a list of genes was generated, which was ordered by increasing significance within this relation. Unexpectedly, only the top three genes evolved by a significant relation to the cell lines’ IC50 for Rpx, these were pyrimidinergic receptor P2Y, G-protein coupled, 6 (P2RY6), gamma-aminobutyric acid (GABA) A receptor, β 3 (GABRB3), and KN motif and ankyrin repeat domains 4 (KANK4). However, the role of these genes within the cell lines’ sensitivity to Rpx was not obvious. To improve this low yield, which presumably resulted from the adjustment for testing 42 000 genes, the following 99 genes of this list were analyzed. An overview of these genes is given in Table 1. There were 14 genes with unknown function, and 62 genes with unknown relation to the cell lines’ drug sensitivity. The remaining 23 genes could be assigned to groups with apparent relation to the cytotoxicity of Rpx. These included (1) inhibition of apoptosis (n = 7, positive relation to IC50), (2) detoxification of/resistance to drugs (n = 6, positive relation to IC50), (3) cell proliferation (n = 3, positive relation to IC50), (4) membrane proteins (n = 3, 2 with positive, 1 with negative relation to IC50), (5) apoptosis induction (n = 1, negative relation to IC50), and (6) tumor suppressor activity (n = 1, negative relation to IC50). In addition, there were 2 genes related to growth arrest (positive relation to IC50) and prevention of apoptosis (negative relation to IC50), but their relation to the cytotoxic effects of Rpx seems implausible. In a second approach the cell lines were grouped into three categories, in line with their sensitivity to Rpx. Group 1 comprised four cell lines (MIAPaCa-2, PANC-89, T3M4, and ASML), which are highly sensitive to Rpx. Group 2 consisted of 7 cell lines (PANC-1, Suit2–007, Suit2–013, AsPC-1, DanG, CFPAC1, and Patu390) that showed a moderate sensitivity

Figure  2. Antineoplastic activity of riproximin in Suit2–007 PDAC rat liver metastasis. (A) Detection of ASML cells after intra-portal implantation into the liver of BDX rats by bioluminescence imaging after one and three weeks, respectively. The light emission was based on luciferase activity of ASMLGFP-LUC cells. The rats were treated with either 100 or 150 ng Rpx /rat (500 µg/kg and 750 µg/kg). (B) Mean tumor weight of BDX rats after intravenous administration of Rpx (500 µg/kg or 750 µg/kg) in comparison to untreated control. *P = 0.001;*P < 0.0001.

CD120b/TNFR2) were concentration- and time-dependently increased, as was their ligand, tumor necrosis factor (TNF/TNFα). Concomitantly, the tumor necrosis factor receptor superfamily members 10a and 10b (TNFRSF10a/CD261/DR4/ APO2/ TRAILR1 and TNFRSF10b/CD262/DR5/Killer) increased similar to FAS, but more distinctly. However, their ligand (tumor necrosis factor superfamily member 10; TRAIL/APO2L) was about 2-fold decreased in expression at all-time points and Rpx concentrations. Somewhat similar to TRAIL, the tumor necrosis factor ligand superfamily member 11 (TNFSF11/CD254/ RANKL) was reduced in expression in response to 50 pM Rpx following 24 h and 72 h. Because of death receptor activation, caspases 8, 9, and 3 were elevated, indicating activation of extrinsic and mitochondrial induction of apoptosis. Connected to the death receptor activation, TNF receptor associated factors 1, 2, 3, and 6 (TRAF1, TRAF2, TRAF3, and TRAF6) were distinctly increased in expression. In accord with these signals, the subunits p105 and p100 of nuclear factor NFκB (NFKB1 and NFKB2) were increased up to 34-fold and 26-fold, respectively.

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to Rpx. The third group included the remaining 4 cell lines (Capan-1, SU.86.86, BxPC3, and Colo357) with the lowest sensitivity to Rpx. Then, the mRNA expression of the grouped cell lines was related to the cytotoxicity of Rpx. Again, the 99 most important genes were selected for analysis. These results are given in the lower part of Table 1. There were 33 genes with unknown function and 59 genes with unknown relation to the cell lines’ drug sensitivity. Only seven genes showed an apparent relation to the cytotoxicity of Rpx. These included (1) inhibition of apoptosis (n = 2, positive relation to IC50), (2) induction of apoptosis (n = 2, negative relation to IC50), (3) cell proliferation (n = 1, positive relation to IC50), (4) detoxification, and (5) DNA repair (n = 1, positive relation to IC50, respectively). There was no overlap of genes between the two selection procedures and the latter method yielded distinctly less plausible associations. Expression of selected genes and sensitivity of PDAC cells For a mechanistic approach, genes were chosen, which were assumed to be related to the cellular uptake of Rpx. Specifically, the CEA-related cell adhesion molecules (CEACAMs) were selected because their gene products are CEA glycoproteins, which are known to carry glycan structures with high affinity to Rpx. The expression pattern of 11 CEACAM genes including 3 splice variants of CEACAM1 is shown in Figure 3A. When the IC50 values of the cell lines were plotted against the respective mean CEACAM expression values, a significant nonlinear regression was obtained (f = a × (1 + x) b, P = 0.0022, Fig. 3B), indicating that cell lines expressing high levels of CEA glycoproteins were more sensitive to Rpx than those with low CEA expression. Expression of apoptosis related genes in response to Rpx A large subgroup of the genes with relation to the cytotoxicity of Rpx was linked to apoptosis induction or inhibition. Therefore, we assumed that the mechanism of action of Rpx involves the modulation of apoptotic signaling chains. This question was followed by exposing Suit2–007 cells to Rpx and determining the modulation of genes, which are implicated in apoptosis induction. Based on the genes of this assay, 33 (41%) and 16 (20%) were modulated at least 5-fold and 10-fold in expression following exposure of Suit-2007 cells to Rpx (Table 2). The range of modulation comprised both, decreases (maximum decrease 20-fold after 72 h of exposure to 50pM Rpx; caspase 5) and increases (maximum increase 198-fold, TNF receptor associated factor 1) in mRNA expression. The selection of low and high Rpx concentrations and the time interval until the cells were harvested allowed further differentiation between these effects. Generally, all death receptor signaling pathways were modulated (see Fig. 4) in a concentration and time dependent manner. This can be exemplified by the cell surface receptor FAS, which showed an about 3-fold reduction in response to 10 pM Rpx after 24 h, but an about 3-fold increased expression in response to 50 pM and this elevated expression increased further (12-fold) after 72 h. The corresponding Fas ligand increased more than 10-fold after 24 h following both concentrations, but decreased to only a 3-fold elevated level after 72 h in response to 50 pM Rpx. Parallel to this, the tumor necrosis factor receptor superfamily members 1A and 1B (TNFRSF1A/CD120a/TNFR1 and TNFRSF1B/

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Approach 2b (n = 99 genes)

Unknown genes (n = 14)

Genes with unknown relation to PDAC (n = 62)

Genes with supposed relation to PDAC (Gene abbreviation/ NCBI reference)

Gene name

Relation to IC50

Supposed function in PDAC

GAS1/NM_002048.2

Growth arrest-specific 1

Negative

Putative tumor suppressor

GSTM1/ NM_000561.3

Cytosolic and membrane-bound forms of glutathione S-transferase mu1

Positive

Detoxification of drugs/toxins

PIGZ/NM_025163

The glycosylphosphatidylinositol (GPI) anchor is a glycolipid

Positive

ER/membrane protein

SEPT4/ NM_001198713.1

Septin 4

Positive

Apoptosis inhibition

NPTX1/ NM_002522.3

Neuronal pentraxin I

Positive

Detoxification (snake toxin taipoxin)

SLCO2B1/ NM_007256.4

Solute carrier organic anion transporter family, member 2B1

Positive

Transmembrane transporter

DCK/NM_000788.2

Deoxycytidine kinase

Positive

Resistance to anticancer chemotherapeutic agents

REXO2/ NM_015523.3

RNA exonuclease 2

Positive

Resistance to cell death through role in DNA repair.

GSTA1/ NM_145740.3

Cytosolic and membrane-bound forms of glutathione S-transferase 1

Positive

Detoxification of drugs/toxins

HSPB3/ NM_006308.2

Heat shock protein 3

Positive

Chaperone/apoptosis inhibition

GSTA4/ XM_005249035.1

Cytosolic and membrane-bound forms of glutathione S-transferase 4

Positive

Detoxification of drugs/toxins

SLC35A5/ NM_017945.3

Solute carrier family 35, member A5

Negative

Transmembrane transporter of nucleotide-sugars

DUSP13/ NM_001007271.1

Dual specificity phosphatase 13

Positive

Cell proliferation and differentiation

IER3/NM_003897.3

Immediate early response 3

Negative

Apoptosis inhibition/drug resistance

ANK3/NM_001149.3

Ankyrin 3

Positive

Proliferation

ALDH1A1/ NM_000689.4

Aldehyde dehydrogenase 1 family, member A1

Positive

Detoxification/alcohol metabolism

KDELR1/ NM_006801.2

KDEL endoplasmatic reticulum protein retention receptor 1

Positive

Retention of ER soluble proteins/ER stress inhibition

RAD21/ NM_006265.2

RAD21 homolog (S. pombe)

Negative

Apoptosis induction

HSPB7/ NM_014424.4

Heat shock protein family, member 7

Positive

Chaperone/apoptosis inhibition

SLC22A17/ NM_020372.3

Solute carrier family 22, member 17

Positive

Apoptosis inhibition

EMOS/ NM_001278182.1

Eomesodermin

Positive

Cell proliferation and differentiation

CEBPA NM_001285829.1

CCAAT/enhancer binding protein (C/EBP)

Positive

Growth arrest in cultured cells

NMRAL1/ NM_020677.3

NmrA-like family domain containing 1

Negative

Preventing apoptosis

a The association between IC50 dosage values and gene expression of cell lines was analyzed using IC50 values as quantitative trait. bThe association between IC50 dosage values and gene expression of cell lines was analyzed by grouping cell lines according to IC50 values into low/medium/high groups.

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Table 1. Overview of genes, for which the expression is related to the respective PDAC cell lines’ sensitivity to riproximin (continued)

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Approach 2b (n = 99 genes)

Unknown genes (n = 14)

Genes with unknown relation to PDAC (n = 62)

Genes with supposed relation to PDAC (Gene abbreviation/ NCBI reference)

Gene name

Relation to IC50

Supposed function in PDAC

PTPRD/ NM_001040712.2

Homo sapiens protein tyrosine phosphatase, receptor type, D

Positive

Cell proliferation and differentiation

SLC22A5 NM_003060.3

Solute carrier family 22 (organic cation/carnitine transporter), member 5

Positive

Elimination of endogenous organic cations, drugs and environmental toxins

CIDEA/ NM_001279.3

Cell death-inducing DFFA-like effector a

Negative

Apoptosis induction

MUC1/ NM_001018016.2

Mucin 1

Positive

Inhibition of apoptosis

CHIA/ NM_001258001.1

Chitinase, acidic

Positive

Preventing apoptosis

PAPD5/ NM_001040284.2

PAP associated domain containing 5

Positive

Involved in DNA repair

The association between IC50 dosage values and gene expression of cell lines was analyzed using IC50 values as quantitative trait. bThe association between IC50 dosage values and gene expression of cell lines was analyzed by grouping cell lines according to IC50 values into low/medium/high groups. a

The increase in NFκB signaling, on the other hand, was associated with the distinctly increased expression of the V-rel reticuloendotheliosis viral oncogene homolog (REL) as well as its family members RELA and RELB (Table 2). FACS analysis Suit2–007 cells were exposed to Rpx (5–50 pM) and living (propidium iodide negative) cells were analyzed for phosphatidylserine expression on the cells’ surface as indicator of apoptosis. The ratio of phosphatidylserine positive cells increased 10-fold from 1.4% (control) to 14% after exposure to Rpx for 72 h, indicating a considerable fraction of cells undergoing apoptosis. Combination effect of riproximin and TRAIL To possibly exploit the modulation of death receptor signaling pathways for an increased cytotoxic effect, Rpx was combined with TRAIL in ASML and Suit2–007 cells. In ASML cells, TRAIL alone caused 50% to 70% cell growth inhibition at 1 to 100 nM concentrations, and in Suit2–007 cells there was 20% cell growth inhibition at a 10 nM concentration (Figs. 5 and 6). The combination of these concentrations of TRAIL with Rpx was synergistically active. Compared with an expected additive effect of the two agents, the observed effect was more than 6-fold higher (P < 0.01).

Discussion PDAC is one of the most aggressive cancers, which is characterized by resistance against current antineoplastic chemotherapy. Only a distinct selection (ca. 5%) of all cytotoxic drugs is clinically used, but a minority of these, again, is licensed for the treatment of PDAC.18 Thus, there is an insistent necessity for new drugs and therapeutic strategies to improve the survival

of patients with advanced PDAC. In addition, the development of suited in vivo models with distinct patho-physiological and clinical relevance to PDAC is expected to aid in finding such anticancer agents. In this study, a panel of PDAC cell lines was used for investigating the antineoplastic activity of Rpx in vitro and in a new PDAC liver metastasis rat model. Rpx showed clear cytotoxic efficacy in this panel; however, the sensitivity of the cell lines for Rpx varied distinctly, as the IC50 values ranged by a factor of 73 after 48 h of exposure. Comparable variation in sensitivity was reported by Voss et al. for cancer cell lines of different origin. In this study, the IC50 values differed by a factor of 100.1 For assessing the contribution of the transcriptome to this difference in sensitivity, we correlated the PDAC cells’ IC50 values for Rpx with their respective mRNA expression profile. The most significant 99 genes resulting from this relation were categorized for their potential influence on Rpx sensitivity. Based on this analysis, 21 genes were functional in the processes of apoptosis (n = 8), detoxification (n = 6), resistance to drugs (n = 3), and cell proliferation (n = 1). In addition, three genes coded for cell membrane proteins and one for a tumor suppressor. The observed variation in sensitivity may be also related to the affinity of Rpx for certain glyco-structures located on the cell membrane, as analyzed recently. The clustered Tn antigen (repetitive N-acetyl-Dgalactosamine; GalNAc), a cancer-specific O-glycan on mucins, as well as bi- and tri-antennary complex N-glycan structures (NA2/NA3) were recently found to be molecules with potent binding affection to Rpx.5 The latter finding, which includes a high affinity of Rpx for CEA, was basis to relate the expression levels of the CEACAM gene group to the respective cells’ sensitivity toward Rpx. This gene group has been recognized to function in cancer progression, inflammation, angiogenesis,

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Table 1. Overview of genes, for which the expression is related to the respective PDAC cell lines’ sensitivity to riproximin (continued)

and metastasis.19 The CEACAMs 1, 5, and 6 are now considered promising therapeutic targets in pancreatic cancer. Their increased expression is an independent predictor of poor overall survival. Therefore, it is remarkable that the IC50 values of the PDAC cell lines used in this study were negatively related to the mean CEACAM gene expression levels, which indicates that PDACs with high CEACAM expression levels have a higher sensitivity to Rpx than those with low CEACAM expression. The correlation of the PDAC cells’ transcriptome with their sensitivity to Rpx had shown that a majority of genes, which were significantly related and had a plausible function, was related to apoptosis. This prompted further evaluation by analyzing the

respective signaling pathways following exposure of PDAC cells to Rpx. The treatment of Suit2–007 cells with Rpx and subsequent examination of apoptosis related genes’ mRNA expression showed a clear concentration and time dependent modulation of the most important death receptors and their corresponding signaling pathways. These included the increased expression of the cell surface receptors FAS and the tumor necrosis factor receptor superfamily members TNFR1 and TNFR2. In addition, even more increased mRNA expression levels of the tumor necrosis factor receptor superfamily members DR4 and DR5 were recorded. These increases coincided with reduced levels of TRAIL, the corresponding ligand for these receptors, as well as

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Figure 3. Association of riproximin’s cytotoxicity with CEACAM gene expression. (A) mRNA expression (arbitrary units) of 14 CEACAM genes in 14 human PDAC cell lines. (B) Correlation of the IC50 values of riproximin in PDAC cell lines vs. the average mRNA expression level of CEACAM genes. A significant nonlinear regression was obtained for f = a × (1 + x)b, with P = 0.0022.

Table 2. Overview of apoptosis related genes modulated in Suit2–007 PDAC cells in response to riproximin (continued)

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24 h 10 pM

24 h 50 pM

72 h 50 pM

v-Akt murine thymoma viral oncogene homolog 1 (AKT1)

0.60

1.00

2.97

apoptotic peptidase activating factor 1 (APAF1)

1.57

3.66

9.51

apoptosis, caspase activation inhibitor (AVEN)

1.74

2.75

4.44

BCL2-associated agonist of cell death (BAD)

1.47

2.48

3.23

BCL2-associated athanogene (BAG1)

4.32

11.00

11.79

BCL2-antagonist/Killer 1 (BAK1)

4.82

9.32

5.21

BCL2-associated X protein (BAX)

1.82

5.78

8.06

BCL2 binding component 3 BBC3

1.09

2.89

1.74

B-cell CLL/lymphoma 2 (BCL2)

1.34

1.10

5.35

BCL2-like 1 (BCL2L1, BCLXL)

1.53

1.72

1.03

BCL2-like 11 (apoptosis facilitator) (BCL2L11)

-

-

3.68

BCL2-like 13 (apoptosis facilitator) (BCL2L13)

2.27

2.25

1.89

BCL2-like 2 (BCL2L2)

1.39

3.58

2.36

BH3 interacting domain death agonist (BID)

1.29

1.47

0.43

BCL2-interacting killer (apoptosis-Inducing) (BIK)

0.62

0.32

0.54

baculoviral IAP repeat containing 2(BIRC2)

2.17

5.13

25.11

baculoviral IAP repeat containing 3 (BIRC3)

3.94

10.63

5.50

baculoviral IAP repeat containing 5 (BIRC5, Survivin)

7.11

23.75

22.32

BCL2-related ovarian killer (BOK)

0.99

1.03

5.24

carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD)

0.67

0.67

0.75

caspase 1, apoptosis-related cysteine peptidase (CASP1)

1.47

1.43

3.32

caspase 10, apoptosis-related cysteine peptidase (CASP10)

0.40

3.73

0.24

caspase 12, apoptosis-related cysteine peptidase (CASP12)

0.92

3.12

3.29

caspase 2, apoptosis-related cysteine peptidase (CASP2)

1.64

2.62

4.86

caspase 3, apoptosis-related cysteine peptidase (CASP3)

2.69

5.13

3.66

caspase 4, apoptosis-related cysteine peptidase (CASP4)

1.35

4.92

2.20

caspase 5, apoptosis-related cysteine peptidase (CASP5)

0.50

3.05

0.05

caspase 6, apoptosis-related cysteine peptidase (CASP6)

0.57

0.41

1.06

caspase 7, apoptosis-related cysteine peptidase (CASP7)

1.61

1.44

2.22

caspase 8, apoptosis-related cysteine peptidase (CASP8)

1.67

4.76

2.62

caspase 8 associated protein 2 (CASP8AP2)

0.77

2.55

5.24

caspase 9, apoptosis-related cysteine peptidase (CASP9)

1.69

3.53

3.66

CASP8 and FADD-like apoptosis regulator (CFLAR, FLIP)

4.79

7.41

2.99

CASP2 and RIPK1 domain containing adaptor with death domain (CRADD)

3.63

7.26

4.72

DNA fragmentation factor, 45kDa, α polypeptide (DFFA)

2.99

2.97

2.39

diablo, IAP-binding mitochondrial protein (DIABLO)

2.11

4.08

3.20

endonuclease G (ENDOG)

1.07

0.75

1.06

5.13

8.17

3.97

Fas (TNFRSF6)-associated via death domain (FADD)

Genes were annotated according to GeneCards (http://www.genecards.org/). For evaluation of the Human Apoptosis Panel Kit, the ΔΔCt method was used to calculate the relative ratio vs. control. In short, CP (crossing point) values, indicating the start of linear amplification were determined by the Second Derivative Maximum Method. From seven reference genes, those with most extreme changes following treatment were excluded. The remaining five (actin-β, β-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a) were used to calculate the mean CP of reference genes. The ΔCP from treated and control samples was used to determine the ΔΔCP value, corresponding to the fold change (ratio = 2−ΔΔCP) given in the table.

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b

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Gene annotationa

Table 2. Overview of apoptosis related genes modulated in Suit2–007 PDAC cells in response to riproximin (continued)

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24 h 10 pM

24 h 50 pM

72 h 50 pM

family with sequence similarity 96, member A (FAM96A)

1.24

1.54

3.07

family with sequence similarity 96, member B (FAM96B)

2.17

3.92

2.66

Fas cell surface death receptor (FAS)

0.35

2.77

12.47

Fas Ligand (FASLG)

10.63

10.27

2.73

high mobility group box 1 (HMGB1)

1.04

0.84

1.91

harakiri, BCL2 interacting protein (contains only BH3 domain (HRK)

0.77

0.52

0.73

heat shock protein 90kDa β (Grp94), member 1 (HSP90B1)

0.38

0.60

0.15

HtrA serine peptidase 2 (HTRA2)

1.46

2.43

3.10

leucine-rich repeats and death domain containing (LRDD)

-

-

1.11

Induced myeloid leukemia cell differentiation protein (MCL1)

2.83

3.18

1.40

nuclear factor NF-kappa-B p105 subunit (NFKB1)

3.27

17.27

33.59

nuclear factor NF-kappa-B p100 subunit (NFKB2)

8.00

26.35

18.64

nerve growth factor receptor (NGFR)

2.43

32.90

35.75

phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1)

2.01

6.15

11.08

phosphatase and tensin homolog (PTEN)

1.82

4.53

3.25

V-rel reticuloendotheliosis viral oncogene homolog (REL)

2.93

12.04

13.00

V-rel reticuloendotheliosis viral oncogene homolog A (RELA)

4.96

13.45

10.41

V-rel reticuloendotheliosis viral oncogene homolog B (RELB)

4.96

18.51

8.00

suppressor of cytokine signaling 2 (SOCS2)

9.51

41.36

25.46

suppressor of cytokine signaling 3 (SOC3)

3.25

7.78

7.67

signal transducer and activator of transcription 1, 91kDa (STAT1)

1.37

3.81

2.69

signal transducer and activator of transcription 5A (STAT5A)

0.50

0.92

1.37

signal transducer and activator of transcription 5B (STAT5B)

1.83

3.81

4.82

tumor necrosis factor (TNF)

1.73

2.38

2.39

tumor necrosis factor receptor superfamily, member 10a (TNFRSF10A, DR4)

4.53

7.84

4.17

tumor necrosis factor receptor superfamily, member 10b (TNFRSF10B, DR5)

3.76

6.82

1.78

tumor necrosis factor receptor superfamily, member 10c, decoy without an intracellular domain (TNFRSF10C)

3.07

6.11

6.73

tumor necrosis factor receptor superfamily, member 10d, decoy with truncated (TNFRSF10D)

3.63

8.75

5.58

tumor necrosis factor receptor superfamily, member 11b (TNFRSF11B)

0.11

0.27

0.34

tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A, TNFR1)

1.26

2.27

2.16

tumor necrosis factor receptor superfamily, member 1B (TNFRSF1B, TNFR2)

1.24

1.62

3.51

tumor necrosis factor receptor superfamily, member 21 (TNFRSF21)

1.19

1.82

2.00

tumor necrosis factor superfamily, member 25 (TNFRSF25)

0.53

0.90

1.41

tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10, TRAIL, APO2L)

0.62

0.39

0.55

tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11, RANKL)

2.08

0.53

0.33

tumor protein P53 (TP53)

1.30

2.10

1.61

2.55

2.53

1.62

tumor protein p53 inducible protein 3 (TP53I3)

Genes were annotated according to GeneCards (http://www.genecards.org/). For evaluation of the Human Apoptosis Panel Kit, the ΔΔCt method was used to calculate the relative ratio vs. control. In short, CP (crossing point) values, indicating the start of linear amplification were determined by the Second Derivative Maximum Method. From seven reference genes, those with most extreme changes following treatment were excluded. The remaining five (actin-β, β-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a) were used to calculate the mean CP of reference genes. The ΔCP from treated and control samples was used to determine the ΔΔCP value, corresponding to the fold change (ratio = 2−ΔΔCP) given in the table.

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Gene annotationa

Table 2. Overview of apoptosis related genes modulated in Suit2–007 PDAC cells in response to riproximin (continued)

24 h 10 pM

24 h 50 pM

72 h 50 pM

TNF receptor-associated factor 1 (TRAF1)

42.81

198.09

59.30

TNF Receptor-associated Factor 2 (TRAF2)

4.35

8.46

5.90

TNF Receptor-associated Factor 3 (TRAF3)

2.60

5.66

4.82

TNF Receptor-associated Factor 5 (TRAF5)

1.27

0.99

0.81

TNF Receptor-associated Factor 6, (TRAF6)

4.76

10.78

5.82

1.09

1.42

1.19

TNF Receptor-associated Factor 7, (TRAF7)

Genes were annotated according to GeneCards (http://www.genecards.org/). For evaluation of the Human Apoptosis Panel Kit, the ΔΔCt method was used to calculate the relative ratio vs. control. In short, CP (crossing point) values, indicating the start of linear amplification were determined by the Second Derivative Maximum Method. From seven reference genes, those with most extreme changes following treatment were excluded. The remaining five (actin-β, β-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase 1, ribosomal protein L13a) were used to calculate the mean CP of reference genes. The ΔCP from treated and control samples was used to determine the ΔΔCP value, corresponding to the fold change (ratio = 2−ΔΔCP) given in the table.

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b

of RANKL. Finally, the activation of these death receptor signaling pathways resulted in stimulation of caspases 8, 9, and 3, which mediate extrinsic and mitochondrial apoptosis. Recently, activation of the Fas/ Fas Ligand apoptotic pathway in response to the RIP type II abrin was reported.20 In addition, induction of the mitochondrial-mediated pathways of apoptosis was observed in U937 and NIH 3T3 cells after treatment with the RIPs saporin-6 and ricin, respectively.21,22 These data are in agreement with the findings of our study. Prompted by the downregulation of TRAIL and the upregulation of its receptors DR4/5 in Suit2–007 cells in response to Rpx exposure, we supposed that the sensitivity of these cells to TRAIL would be increased. Figure 4. Schematic overview of the expression of some apoptotic players of Suit2–007 PDAC cells mediTherefore, we treated two PDAC ating extrinsic and mitochondrial apoptosis. The status is shown after 72 h in response to 50 pM Rpx. The gene abbreviations refer to those of Table 2 (highlighted in bold). cell lines (ASML and Suit2–007) with Rpx and TRAIL alone or in combination. The two PDAC cell lines were similarly sensitive to Rpx, but differed in sensitiv- yielded distinctly less activity.23 Interestingly, the 6-fold increase ity toward TRAIL. Exposure to 1 nM TRAIL caused inhibi- in activity corresponds about to the ratio of expression levels tion of proliferation by 50% and 0% in ASML and Suit2–007 observed for the TRAIL receptors and their ligand at 24 h after cells, respectively. Surprisingly, there was no further substantial exposure to 10 pM Rpx. increase in the antineoplastic efficacy of TRAIL at higher conFor testing of the antineoplastic effect in vivo, we established centrations (10 and 100 nM) for ASML cells, while the 10 nM a new in vivo model of pancreatic cancer metastasis to the liver. concentration caused 30% growth inhibition in Suit2–007 cells. This model is based on the rat PDAC cell line ASML, which was Remarkably, combination of TRAIL with a concentration of Rpx injected intra-portally to mimic the natural way of liver metastacausing about 50% inhibition of proliferation was synergistically sis by PDAC cells, as described previously.17,24 The effect of Rpx active in the two cell lines. Compared with an expected additive in this model was significant in terms of tumor growth reduction effect of the two agents, the observed effect was more than 6-fold as shown for colorectal cancer liver metastasis before.1 Future higher in both cell lines (P < 0.01). Other synergistic combina- experiments will concentrate on the putative synergism of Rpx tions with TRAIL in PDAC cell lines, e.g., with gemcitabine, and TRAIL in vivo.

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Gene annotationa

Figure 5. Combination effect of riproximin and TRAIL in rat ASML PDAC cells. (A) Inhibition of ASML cell proliferation after 48 h of monotherapy with riproximin (IC50 concentration) and various concentrations of TRAIL. (B) Expected and observed inhibition (%) of ASML cell proliferation after 48 h of combination treatment with riproximin (IC50 concentration) and TRAIL at different concentrations. *P < 0.001

In conclusion, Rpx showed high, but varying cytotoxicity in a panel of 17 PDAC cell lines. When linking this differential sensitivity to the cells’ gene expression profile, a substantial portion of genes with apparent association to Rpx’s cytotoxicity was related to apoptosis. Another association was found for the expression of CEACAM genes, as the affinity of Rpx for CEA was basis for its increased cytotoxicity in cells with high vs. low CEACAM gene expression. Exposure of PDAC Suit2–007 cells to Rpx caused induction of almost all apoptosis signaling pathways, especially of those initiating from Fas, TNFR1 and 2, and DR4 and 5, as well as down modulation of TRAIL. Apoptosis was induced, as confirmed by flow cytometry. Combination of Rpx with TRAIL resulted in a synergistic cytotoxic effect in human Suit2–007 and rat ASML PDAC cells, as evidenced by a more than 6fold lower tumor cell survival than expected from an additive combination effect. Treatment of BDX rats bearing intra-portally implanted Suit2–007 cells for mimicking PDAC liver metastasis showed a highly significant anticancer effect and indicated a selectivity of Rpx for gastrointestinal cancer metastasis to the liver. These data favor a further evaluation of Rpx as anticancer agent.

Cell culture All pancreatic cancer cell lines used are shown in Table S1. The cells were maintained under standard culture conditions in an incubator (37 °C in humidified air with 5% CO2) in their respective medium enriched with fetal bovine serum and l-glutamine. For keeping the cells in logarithmic growth, they were propagated 1–3 times per week, depending on their growth rate. MTT assay For determining the proliferation rate of PDAC cell lines and riproximin’s effect on their propagation, cells were seeded at densities of 1 × 103 –16 × 103 cells/mL (100 µL medium per well) into 3 to 8 wells of 96 well-plates (flat bottom, Becton Dickinson). After 24 h the cells were exposed to Rpx (0–100 pM final concentration) by adding 100 µL full medium containing appropriate Rpx amounts and further grown for 24, 48, 72, and 96 h. After these periods, 10 µL MTT (3-[4.5-dimethylthiazol-2-yl]2.5-diphenyl tetrazolium bromide, Serva) solution (10 mg/mL) was added and following an incubation period of 3 h at 37 °C, the medium was discarded and the cells were lysed by adding 200 µL per well acidified 2-propanol (0.04 N HCl). After all formazan crystals had been carefully dissolved, the absorption was measured at 540 nm (reference filter of 690 nm) in an automated microtiter plate spectrophotometer (Anthos Mikrosysteme). The absorption of exposed cells was given in percent of untreated control cells. For combination effects of Rpx with TRAIL, ASML or Suit2– 007 cells were seeded at appropriate densities (4 × 103 for ASML and 3 × 103 for Suit2–007). After 24 h the cells were exposed to Rpx (0–10 pM final concentration), TRAIL (0–100 nM), or both agents. The experiments were terminated after 48 h and evaluated as described above. Reagents and chemicals Riproximin was purified from Ximenia americana kernels according to a described method.25 For the in vitro and in vivo experiments, Rpx was diluted in full medium or in sterile physiological saline containing 5% albumin, respectively. Animals All animal experiments were performed in accordance with the Regierungspräsidium Karlsruhe, which as Institutional Animal Care and Use Committee (IACUC) approved the animal ethics of this study for the German Cancer Research Center (DKFZ). According to this permit, the animals were randomly allocated to treatment and control groups and they were humanely euthanized after 4 wk at the end of the experiment or when they met certain clinical criteria indicating a moribund status. The criteria used to determine when the animals should be humanely euthanized included a weight loss of more than 10%, pale skin color indicating anemia, icterus, as well as pain or reduced general conditions as indicated by reluctance to move, abnormal posturing, and decreased appetite. All animals were daily monitored for their condition. For sacrifice, a narcosis with isofluorane followed by CO2 was used. Steps taken to minimize suffering of the animals, included analgesics (metamizole) administered post-surgically for up to 3 d and anesthetics (isofluorane) for inhalation

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Material and Methods

anesthesia during surgery. As appropriate, the German guidelines, which are similar to the ARRIVE guidelines for reporting animal studies, were followed. BDX rats of both sexes were used for the in vivo experiments. Since this strain is not commercially available, they were bred in the central animal facility of the DKFZ and obtained at an age of 5–7 wk and a corresponding body weight of 120–160 g. They were kept under specific pathogen free (SPF) conditions in Macrolon-III-cages of a ventilated rack (Ventirack, UNO Roestvaststaal B.V.) providing a 50-fold exchange of filtered air per hour as well as positive air pressure inside the cages. Constant room temperature (22 ± 1 °C), air humidity (50 ± 10%) and dark–light rhythm (12 h) were maintained throughout. The animals had free access to autoclaved water and standard laboratory diet. An acclimatization period of 7 d was adhered to before starting any experiments. Tumor cell transplantation Logarithmically growing ASML GFP-LUC cells were trypsinized and suspended (2 × 106 cells) in 0.25 mL PBS (phosphate buffered saline without calcium and magnesium ions) and 0.15 mL matrigel (extract of the Engelbreth-Holm-Swarm-mouse tumor; Biomatrix EHS solution, Serva). This suspension was stored on ice until injection. For tumor cell transplantation the rats were anesthetized with isoflurane at 1.5 vol% together with 0.5 L/min oxygen and 1 L/min nitrous oxide. After a median laparotomy, the cecum was exteriorized onto a compress moistened with sterile physiological saline and a mesocolic vein was isolated from mesenteric fat. Under microscopic control the tumor cell suspension was injected into this vessel with a 28-gauge needle. Thereafter the vessel was compressed with two cotton swabs for a period of 1–2 min to prevent bleeding; the cecum was moved back into the abdomen; the musculature was sutured (4–0 vicryl, Ethicon GmbH) and the skin was closed with metal clips. Each of the three experimental groups consisted of 15 tumor bearing rats. In vivo imaging Live animal bioluminescence imaging was performed using the IVIS100 imaging system (Xenogen Corp.). Prior to imaging, the animals were injected intraperitoneally with the substrate D-Luciferin

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Figure  6. Combination effect of riproximin and TRAIL in human Suit2–007 PDAC cells. (A) Inhibition of Suit2–007 cell proliferation after 48 h of monotherapy with riproximin (5 pM or 10 pM) and various concentrations of TRAIL. (B) Expected and observed inhibition (%) of Suit2–007 cell proliferation after 48 h of combination treatment with riproximin (5 pM) and TRAIL at different concentrations. (C) Expected and observed inhibition (%) of Suit2–007 cell proliferation after 48 h of combination treatment with riproximin (10 pM) and TRAIL at different concentrations. *P < 0.001

The cRNA was column purified according to the Total Prep RNA Amplification Kit, and eluted in 80 µL of water. Quality of cRNA was controlled using the RNA Nano Chip Assay on an Agilent 2100 bio-analyzer and spectrophotometrically quantified (NanoDrop). Hybridization was performed at 58 °C, in GEX-HCB buffer (Illumina Inc.) at a concentration of 100 ng cRNA/µL, unsealed in a wet chamber for 20 h. Spike-in controls for low, medium and highly abundant RNAs were added, as well as mismatch control and biotinylation control oligonucleotides. Microarrays were washed once in High Temp Wash buffer (Illumina Inc.) at 55 °C and then twice in E1BC buffer (Illumina Inc.) at room temperature for 5 min (and between washed with ethanol at room temperature). After blocking for 5 min in 4 mL of 1% (wt/ vol) Blocker Casein in phosphate-buffered saline Hammarsten grade (Pierce Biotechnology, Inc.), array signals were developed by a 10 min incubation in 2 mL of 1 µg/mL Cy3-streptavidin (Amersham Biosciences) solution and 1% blocking solution. After a final wash in E1BC, the arrays were dried and scanned. Scanning and data analysis Microarray scanning was done using an iScan array scanner. Data extraction was done for all beads individually, and outliers were removed when >2.5 MAD (median absolute deviation). All remaining data points were used for the calculation of the mean average signal for a given probe, and standard deviation for each probe was calculated. Statistical analysis Quantile-normalized Illumina array expression data were log2 transformed. The association between IC50 dosage values and gene expression of cell lines was analyzed (1) using IC50 values as quantitative trait, and grouping cell lines according to IC50 values into (2) low/medium/high groups. Differentially expressed transcripts were identified using the empirical Bayes approach,28 based on moderated t-statistics/F-statistics as implemented in the Bioconductor package limma.29 All P values were adjusted for multiple testing using Benjamini–Hochberg correction to control the false discovery rate. All P values are two-sided. Adjusted P values below 0.05 were considered statistically significant. All analyses were performed using software R.30 Nonlinear regression (power; 2 parameter modified II) between IC50 values and the sum of CEA genes’ (n = 14) mRNA expression (arbitrary units) was calculated by using the Sigma plot version 12.3 statistic package. The differences in tumor growth were determined by multivariate analysis using the multiple comparisons option of the Kruskal Wallis test. A two-sided P value < 0.05 was considered significant. Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed. Supplemental Materials

Supplemental materials may be found here: www.landesbioscience.com/journals/cbt/article/29503/

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(Synchem Corp.) at a dose of 10 mg/animal and anesthetized with isoflurane/oxygene via the XGI-8 Anesthesia System (Xenogen Corp.), The images obtained by the IVIS 100 camera were subsequently analyzed using the Living Image v2.5 software provided by Xenogen Corp. In vivo experiments For determining the effect of riproximin in a rat liver metastasis model, the ASML-rat model was established in BDX rats. Tumor-bearing rats (three groups with 15 rats, respectively) were treated intravenously twice a week for up to 3 wk with 2 concentrations of riproximin (100 or 150 ng/rat) in comparison to an untreated control group. The dosages used for treatment were based on earlier studies. To observe the tumor development, the animals were imaged according to the previously described protocol (see in vivo imaging). Three to four weeks later, the experiment was terminated, the animals euthanized, the liver of the animals was removed and weighed. qRT-PCR Single-stranded cDNA was generated from total RNA using the Omniscript RT Kit (Qiagen) according to the manufacturer’s protocol. Subsequent quantitative PCR reactions were performed with the Real Time ready Human Apoptosis Panel, 96 (Roche), which is based on the Universal Probe Library from the same company. The kit was followed according to the instructions of the manufacturer and the 96 well plates were run on a Lightcycler480 from Roche. The panel consists of 84 pretested ready to use qPCR assays. Each assay includes the appropriate primers and a universal probe library probe that contains locked nucleic acid. For evaluation, the apoptosis related genes as well as the six household genes determined in control and riproximin treated samples were related and checked for variations in expression after 24 and 72 h. All genes that were modulated in expression more than 2-fold are listed in Table 2. FACS analysis Suit2–007 cells were exposed to Rpx (0, 5, 10, 25, and 50 pM), harvested after 48 and 72 h and stained with FITC coupled annnexin V (eBioscience) as well as propidium iodide according to the manufacturer’s protocol. Staining intensity as well as the percentage of propidium iodide negative (living) cells presenting phosphatidylserine on their membrane was determined by a FACSCalibur (Becton Dickinson). Microarray Probe Labeling and Illumina Sentrix BeadChip array Hybridization was performed as described before.26 Biotinlabeled cRNA samples for hybridization on Illumina Human Sentrix-12 BeadChip arrays (Illumina, Inc.) were prepared according to Illumina’s recommended sample labeling procedure based on the modified Eberwine protocol.27 In brief, 200 ng total RNA was used for cDNA (cDNA) synthesis, followed by an amplification/labeling step (in vitro transcription) to synthesize biotin-labeled cRNA according to the Illumina® Total Prep™ RNA Amplification Kit (Life Technologies). Biotin-16-UTP was purchased from Roche Applied Science.

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1.

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