Hindawi Publishing Corporation Evidence-Based Complementary and Alternative Medicine Volume 2012, Article ID 605154, 11 pages doi:10.

1155/2012/605154

Research Article Involvement of Prohibitin Upregulation in Abrin-Triggered Apoptosis

Yu-Huei Liu,1, 2, 3 Konan Peck,4 and Jung-Yaw Lin1
1 Institute

of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan Institute of Integrated Medicine of Chinese Medicine, China Medical University, Taichung 404, Taiwan 3 Department of Medical Genetics and Medical Research, China Medical University Hospital, Taichung 404, Taiwan 4 Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan
2 Graduate

Correspondence should be addressed to Jung-Yaw Lin, [email protected] Received 20 May 2011; Accepted 18 July 2011 Academic Editor: Senthamil R. Selvan Copyright 2012 Yu-Huei Liu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abrin (ABR), a protein puried from the seeds of Abrus precatorius, induces apoptosis in various types of cancer cells. However, the detailed mechanism remains largely uncharacterized. By using a cDNA microarray platform, we determined that prohibitin (PHB), a tumor suppressor protein, is signicantly upregulated in ABR-triggered apoptosis. ABR-induced upregulation of PHB is mediated by the stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) pathway, as demonstrated by chemical inhibitors. In addition, ABR signicantly induced the expression of Bax as well as the activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) in Jurkat T cells, whereas the reduction of PHB by specic RNA interference delayed ABR-triggered apoptosis through the proapoptotic genes examined. Moreover, our results also indicated that nuclear translocation of the PHB-p53 complex may play a role in the transcription of Bax. Collectively, our data show that PHB plays a role in ABR-induced apoptosis, which may be helpful for the development of diagnostic or therapeutic agents.

1. Introduction
Abrin (ABR), puried from the seeds of Abrus precatorius, belongs to the family of type II ribosome-inactivating proteins (RIPs) that contain 2 subunits. These include a toxic A chain with RNA N-glycosidase activity and a galactosebinding B chain with lectin activity [1]. Like ricin from Ricinus communis, the A chain of ABR functions via the inhibition of protein biosynthesis through depurination of a single adenine residue (A4324 ) of the 28S ribosomal RNA [2, 3]. In contrast, the B chain of ABR functions by interacting with the galactose moiety of glycoproteins or glycolipids on the cell membrane and is internalized into cells through receptor-mediated endocytosis. Several reports have documented that ABR is mitogenic [4], antifertility [5], antitumoral [6, 7], and immunopotentiating [812] agent. In addition to its ability to inhibit protein synthesis, ABR is believed to adopt alternative mechanisms to trigger cell apoptosis [13]; despite this, it is relatively less toxic to normal cells than to cancer cells [10, 14]. Our previous

studies have implied that apoptosis induced by ABR could be partially independent of its RNA N-glycosidase activity and instead be mediated by its binding and the decrease of antioxidant protein-1 (AOP-1), increase of reactive oxygen species production, and release of cytochrome c into the cytosol [15]. Prohibitin (PHB) is localized on the cell membrane, mitochondria, and nucleus; this localization may play a pivotal role in its regulation of cell-cycle progression by the inhibition of DNA replication in multiple cell types [16]. The protumorigenic versus antitumorigenic role of PHB in cancer cells remains controversial. An oncogenic role has been identied for PHB in dierent kinds of cancer cells, including those of the breast [17], bladder [18], gastric [19], ovary [20], and prostate [21], whereas PHBs role as a tumor suppressor has been demonstrated in esophageal squamous cell carcinoma [2226]. These opposing eects of PHB in cancer may be due to 2 possible mechanisms. One is a polymorphism in PHB [27]. The other involves its

2 subcellular localization; increased levels of PHB on the cell membrane facilitates tumorigenesis through its interaction with c-Raf induced by the Ras oncogene [28], whereas increased levels of PHB in the nucleus induces apoptosis by increasing the transcriptional activity of p53 and its translocation to the cytoplasm [29]. In order to understand the genetic basis of the apoptotic signaling exerted by ABR, a microarray platform was used to investigate the expression proles of genes in Jurkat T cells after ABR exposure. Among the genes identied, PHB was signicantly upregulated; however, it has yet to be determined whether PHB plays a role in ABR-triggered apoptosis. Here, we report that overexpression of PHB is involved in ABR-triggered Jurkat T cell apoptosis. Upregulation of PHB through the JNK/SAPK pathway activated the pro-apoptotic gene Bax via the accumulation and translocation of the PHB-p53 complex to the cytoplasm. The elucidation of the changes in gene expression and the cellular mechanisms of the response to ABR exposure may be helpful for the development of diagnostic or therapeutic agents.

Evidence-Based Complementary and Alternative Medicine biotin or digoxigenin for membrane hybridization, dualcolor detection, and image analysis as described previously [31]. 2.5. Antibodies and Chemical Inhibitors. An antibody specic to PHB was purchased from Lab Vision Corporation. An antibody specic to p53 was obtained from Santa Cruz Biotechnology. Antibodies specic to cleaved-caspase3, and cleaved-poly(ADP-ribose) polymerase (PARP) were purchased from Cell Signaling. Antibodies against Bax and actin were obtained from Chemicon. The chemical inhibitors PD 98059, SB 203580, and SP600125 were purchased from Sigma. 2.6. Immunoblotting. Total cell lysates were collected in lysis buer (50 mM HEPES-KOH, pH 7.5, 1% Triton X-100, 150 mM NaCl, and protease inhibitor cocktail (Roche)). The extracts were centrifuged at 14000 rpm for 20 min, and then the clear supernatant was separated by using 10% SDS-PAGE. After transferred, the separated proteins to a polyvinyldene uoride (PVDF) membrane (ImmunobilonP, 0.45 mm; Millipore, Billerica, Mass, USA) by using the NA-1512 semi-dry transfer apparatus (NIHON EIDO), the membranes were blocked with 5% skim milk in trisbuered saline containing 1% Tween 20 (TBST, pH 7.4) at room temperature for 30 min and then incubated overnight at 4 C with primary antibodies. The membranes were washed 4 times with TBST for 10 min each at room temperature and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The membranes were then washed 4 times with TBST. The proteins were visualized using the SuperSignal West Femto Chemiluminescent Kit (Thermo Scientic) and exposed to an X-ray lm (Kodak). The Image J program (http://rsb.info.nih.gov/) was used for quantization the expression fold. For western blot analysis, the fold increase of the indicated proteins was determined by normalizing to corresponding actin expression. For IPwestern analysis, the densitometry readings of the bands were normalized to control. 2.7. Enzyme-Linked Immunosorbent Assay (ELISA) for Phosphor MAPK Detection. Cells were treated with or without ABR after pretreating with the indicated inhibitors or vehicle (DMSO) alone. After the indicated period of time, cells were harvested and lysed as above. The PathScan MAP Kinase Multi-Target Sandwich ELISA kit was used to determine phosphor ERK, -p38, and-JNK/SAPK levels according to manufacturers instruction (Cell Signaling). 2.8. Short Interfering RNA . Short interfering RNAs (siRNAs) against PHB (sc-37629) and the negative control siRNA (sc-37007) were obtained from Santa Cruz Biotechnology. A total of 2 105 cells were plated in a 6-well plate for 24 h, and siRNA transfection was carried out using the Lipofectamine 2000 kit according to the manufacturers instructions (Invitrogen).

2. Materials and Methods

2.1. Isolation of ABR. ABR was isolated from seeds of the red variety of A. precatorius using Sepharose 6B anity column chromatography and puried as described in a previous study [30]. The purity and molecular weight of ABR protein were conrmed by Coomassie blue staining (data not shown). The stock protein solution was diluted with phosphate-buered saline (PBS, pH 7.2) to a concentration of 100 M. 2.2. Cells and Culture Conditions. The human Jurkat T leukemia cancer cell line was obtained from American Type Culture Collection and maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin. Cells were grown in suspension in a 5% CO2 humidied atmosphere at 37 C. 2.3. Cell Proliferation Assay. Cells were seeded in 96-well plates at a density of 5 103 cells per well and were treated with (various doses) or without ABR. At the indicated times, viable cells were analyzed by measuring the conversion of the tetrazolium salt 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H5-tetrazolio]-1,3-benzene disulfonate (WST-1) to formazan. The formazan dye produced by metabolically active cells was measured with a scanning multiwell spectrophotometer after 4 h of incubation, according to the manufacturers instructions. 2.4. The cDNA Microarray System. The gene expression patterns regulated by ABR were analyzed with a previously established cDNA microarray platform (9600 probes) [31]. The mRNA from cells with or without ABR treatment was extracted using an Oligotex-dT column (Qiagen). A 2 mg quantity of each mRNA sample was labeled with

Evidence-Based Complementary and Alternative Medicine 2.9. Terminal Deoxynucleotidyl Transferase-Catalyzed Deoxyuridine Triphosphate (dUTP)-Nick End Labeling (TUNEL) Method [15]. Apoptotic cell death was examined by TUNEL method as manufactorys suggestion (Roche Molecular Biochemicals). Each sample with 1 104 events was analyzed with a Becton-Dickinson FACSCalibur, and the distribution of cells was determined. 2.10. Chromatin Immunoprecipitation (ChIP) Assays. Cells treated with or without ABR for the indicated time periods were examined. After xing the protein-DNA complex using formaldehyde (1% nal concentration) at room temperature for 10 min, the reaction was stopped with glycine. After washed and lysed the cells, the lysates were sonicated and centrifuged, and the supernatants were used for immunoprecipitation of Bax with PHB antibody or control IgG. Antibody-bound protein/DNA complexes were precipitated and eluted in 300 L of elution buer (1% SDS, 50 mM NaHCO3 ). Cross-linking was reversed by heating at 65 C for 4 h. The DNA was resuspended in 200 L of distilled water and treated with 30 g of proteinase K at 37 C for 1 h, followed by phenol/chloroform extraction and ethanol precipitation. PCR was conducted using 100 ng of DNA as the template. The following PCR primers were used for the Bax promoter: forward primer 5 -CCGGGAATTCCAGACTGCA-3 and reverse primer 5 -AGCTCTCCCCAGCGCAGAA-3 . Each band was quantitatively determined using the Image J program (http://rsb.info.nih.gov/). The densitometry readings of the bands were normalized to the input. 2.11. Statistical Analysis. SPSS 12.0 for Windows (SPSS Inc.) was used to analyze the data. A two-tailed pairedsamples Students t-test was used for statistical analysis of the comparative data from the two groups. P-value <0.05 values were considered statistically signicant.

3 T cells were rst treated with ABR (0.110 nM). As shown in Figure 1(b), ABR signicantly increased the expression of PHB protein after treatment for 9 h. Cells were further treated with 1 nM ABR for dierent time periods. An initial increase of PHB protein was observed at the 3 h time point and was sustained for up to 18 h after ABR treatment (Figure 1(c)). To determine whether PHB upregulation due to ABR is because of increased transcription or increased RNA stability, the RNA synthesis inhibitor actinomycin D or the protein synthesis inhibitor cyclohexamide was preincubated with cells for 1 h before ABR was added. The results show that not only actinomycin D but also cyclohexamide signicantly diminished ABR-induced PHB upregulation (Figure 1(d)). This nding suggests that ABRinduced PHB upregulation requires de novo RNA synthesis. 3.2. ABR Upregulates PHB Expression through the SAPK/JNK Pathway. To explore which of the signaling pathways are required for ABR-induced upregulation of the PHB gene, several specic chemical inhibitors were used. The eect of these inhibitors was examined using an ELISA-based detection system (Figure 2(a)). Jurkat T cells were pretreated with PD98059 (PD, MEK inhibitor; 20 M), SB203580 (SB, p38 MAPK inhibitor; 20 M), or SP600125 (SP, JNK/SAPK inhibitor; 30 M) for 1 h, followed by treatment with ABR for the time indicated; total protein was used to determine PHB expression. SP signicantly reduced the ABR-induced PHB expression, whereas the 2 other kinase inhibitors PD and SB rarely aected on the upregulation of PHB (Figure 2(b)). These results suggest the possible involvement of JNK/SAPK, but not of ERK1/2 or p38 MAPK, in the regulation of PHB expression in Jurkat T cells treated with ABR. 3.3. PHB Is Involved in ABR-Induced Cell Apoptosis. Since ABR induced the expression of PHB, a tumor suppressor gene that may induce cell apoptosis by arresting the cell cycle at the G1/S phase, we focused on determining whether PHB participates in the apoptotic signaling triggered by ABR. Apoptosis induced by ABR (0.1 and 1 nM) was investigated using TUNEL method. As shown in Figure 3(a), group 1 and 2, the maximal apoptotic response was achieved 18 h after treating cells with ABR (77.7% apoptosis after 1 nM ABR treatment versus 38.5% apoptosis after 0.1 nM ABR treatment; P < 0.001). Although the specic PHB RNA interference (PHB siRNA) did signicantly enhance cell apoptosis (PHB siRNA induces 22.8% apoptosis versus control siRNA induces 7.4% apoptosis, P < 0.001; Figure 3(a) groups 1 and 3), knockdown PHB reduced ABR-induced apoptosis (PHB siRNA reduced 1 nM ABR-induced apoptosis by 9.7%, P < 0.001, and PHB siRNA reduced 0.1 nM ABR-induced apoptosis by 16.6%, P < 0.001; Figure 3(a) groups 2 and 4). Apoptosis-related genes including Bax, caspase-3, and PARP were also examined. ABR signicantly induced the expression of Bax (6.3-fold; P < 0.001) as well as the activation of caspase-3 and PARP in the Jurkat T cells (Figure 3(b), lanes 1 and 2). However, the activation was reduced when the PHB expression reduced (Figure 3(b),

3. Results
3.1. ABR Induces Upregulation of PHB in Human Jurkat T Cells. To evaluate the eect of ABR on leukemia cells in vitro, Jurkat T leukemia cells were exposed to 0.01100 nM of ABR for 24 h. The cell viability was then determined by WST-1 assay. As shown in Figure 1(a), the growth of Jurkat T cells was reduced by ABR in a dose-dependent manner. The value of the 50% cytotoxic concentration (CC50 ) for the 24 h treatment was determined for the water fraction to be 0.32 0.06 nM. The data are represented as mean SD from 3 independent experiments. Microarray analysis was used to identify novel candidates that are dierentially expressed after 1 nM ABR treatment for 3 h. A total of 128 genes, out of the 9600 probes tested, were signicantly altered by >1.5-fold in response to ABR (P < 0.05). The top 10 signicant up-/down-regulated genes are listed in Table 1, sorted by fold increase or decrease. PHB, a signicantly upregulated gene with diverse cellular functions, was selected for further investigation. To explore the potential role of PHB in ABR-treated apoptosis, Jurkat

4
120 Cell survival (% of control ) 100 80 60 40

Evidence-Based Complementary and Alternative Medicine

ABR Dose PHB 0 0.1 1 10 (nM)

20 0 0 0.01 0.1
(a)

1 1 10 100 Concentration (nM) Actin

10.5

12.7

Western blot

(b)

ActD ABR ( 1 nM) Time PHB 1 Actin

(c)

CHX 12 18 (h) ABR ( 1 nM) PHB

+ +

5.8

7.9

12.4

13.6

16.8

Western blot Actin

4.9

0.4

0.7

0.3

1.2

Western blot

(d)

Figure 1: Abrin (ABR) upregulates prohibitin (PHB) expression through transcriptional regulation in Jurkat T cells. (a) ABR-induced cytotoxic activity in a dose-dependent manner in Jurkat T cells after 24 h treatment. The data are represented as mean SD from 3 independent experiments. (b) ABR (0.110 nM) signicantly increased the expression of PHB after treatment for 9 h. (c) ABR (1 nM)induced upregulation of PHB in a time-dependent manner. (d) ABR-induced PHB upregulation requires de novo RNA synthesis.

lanes 3 and 4). The data showed that PHB is involved in ABRtriggered apoptosis.

ABR induces the formation of the PHB-p53 complex in the nucleus, which enhances the transcriptional activity of p53 on Bax following apoptosis.

3.4. Upregulation of Human Bax Expression through Translocation of the PHB-p53 Complex from the Cytoplasm to the Nucleus. The previously described results raised the possibility that PHB may be involved in the apoptotic processes triggered by ABR. On the other hand, increased levels of PHB in the nucleus may interact with the tumor suppressor protein p53 by which it exerts its apoptotic eect. Therefore, we attempted to determine whether there was an interaction between PHB and p53. As shown in Figure 4(a), PHB was translocated from the cytoplasm to the nucleus after ABR treatment for 6 h. Furthermore, a 1.3-, 1.4-, and 3.2-fold increase in p53 and a 1.1-, 1.3-, and 3.1-fold increase in the interaction with PHB were observed when cells were treated with ABR for 3, 6, and 9 h, respectively (Figure 4(b)). These results indicated that ABR may induce a physical interaction between PHB and p53 in the early stage of ABR-induced cell apoptosis. Since Bax is known to be one of the transcriptional regulation targets for PHB and p53, a ChIP assay was performed by using specic primers to amplify a potential p53-binding region in Bax. As shown in Figure 4(c), p53 was recruited to the promoter regions of Bax in a time-dependent manner. These results suggest that

4. Discussion
Studies have shown that some proteins, including ABR, ricin, modeccin, diphtheria toxin, shiga toxin, and pseudomonas toxin, are apoptosis inducers [3234]. Although ABR has been clearly identied as an inducer of apoptotic cell death by activating caspase-3 in several kinds of cancer cells [15, 35 38], the mechanisms of its involvement in cell apoptosis remain to be investigated. In this study, PHB is shown to be upregulated in a dose-dependent manner during ABR treatment and might play a potent role in ABR-triggered apoptosis by enhancing the activity and expression of p53. To the best of our knowledge, this is the rst study to explain and demonstrate the role of PHB in ABR-induced apoptosis in human leukemia cells. The potential clinical applications of ABR may involve the enhancement of drug targeting as well as a decrease in side eects on noncancerous cells. ABR is a RIP, which induces a shutdown of protein synthesis in target cells [33, 39]. However, previous reports also showed that the apoptosis-related protein Bax can be upregulated by ABR [40, 41]. Our results also indicated an overexpression of PHB and p53. One explanation is

Table 1: Top 10 up-/downregulated genes changing in response to abrin exposure arranged by fold change. Gene common name, description, and gene ontology classication (where known) are listed. Fold change GO biological process Upregulation Transcription, DNA dependent Transcription activator activity Catalytic activity Binding to DNA and protein Hormone activity Transcription coactivator/corepressor activity Transmembrane transport GO molecular function 12.9 3.0 2.5 2.4 2.3 Transcription, DNA dependent GO cellular process Nucleus Plasma membrane Nucleus Extracellular Nucleus

Unigene number

Common name

Description

Hs.326035

EGR1

Hs.502769

SLC3A2

Hs.2178

HIST2H2BE

Evidence-Based Complementary and Alternative Medicine

Hs.82963

GNRH1

Early growth response 1 Solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 Histone cluster 2, H2be Gonadotropin-releasing hormone 1 (luteinizing-releasing hormone) Nucleosome assembly Multicellular organismal development

Hs.467408

TRIM28

Tripartite motif-containing 28

Hs.514303

PHB

Prohibitin

2.3

Transcription activator/repressor activity

Cytoplasma, plasma membrane, mitochondria, nucleus

Hs.534404 Hs.5120 Hs.226390 Hs.202207 2.1

RPL10 DYNLL1 RRM2 OSCP1

2.2 2.2 2.1 2.1

Negative regulation of cell proliferation, gene-specic transcription from RNA polymerase II promoter by competitive promoter binding; regulation of apoptosis; signal transduction Translation Induction of apoptosis DNA replication Transport Cell projection organization

Structural constituent of ribosome Motor activity Oxidoreductase activity Hydrolase activity

Cytosol Cytosol Cytosol Plasma membrane Cytoplasma

Hs.25524

PTPN23

Ribosomal protein L10 Dynein, light chain, LC8-type 1 Ribonucleotide reductase M2 Organic solute carrier partner 1 Protein tyrosine phosphatase, nonreceptor type 23

Table 1: Continued. Fold change GO biological process GO molecular function

1.8

Unigene number Downregulation Cellular protein metabolic process Structural molecule activity Translation elongation factor activity Protein binding Protein binding Structural constituent of ribosome Structural constituent of ribosome Protein binding Translation elongation factor activity Structural constituent of cytoskeleton Unfolded protein binding Translation Cellular component movement Positive regulation of apoptosis Translation Translation Negative regulation of cell growth Translational elongation

Common name

Description

GO cellular process

Hs.725987
1.6 1.6 1.5 1.5 1.5 1.5 1.5 1.5 1.5

TUBA1C

Tubulin, alpha 1c

Cytosol Cytosol Cytosol Cytosol Cytosol Cytosol Nucleus, cytosol Cytosol Cytosol Cytosol

Hs.535192

EEF1A1

Hs.514581

ACTG1

Hs.5662

GNB2L1

Hs.534346 Hs.433427

RPS7 RPS17

Hs.5662

GNB2L1

Hs.444467

EEF1G

Hs.514581

ACTG1

Eukaryotic translation elongation factor 1 alpha 1 Actin, gamma 1 Guanine nucleotide-binding protein (G protein), beta polypeptide 2-like 1 Ribosomal protein S7 Ribosomal protein S17 Guanine nucleotide-binding protein (G protein), beta polypeptide 2-like 1 Eukaryotic translation elongation factor 1 gamma Actin, gamma 1 Cellular component movement Regulation of type I interferon-mediated signaling pathway

Evidence-Based Complementary and Alternative Medicine

Hs.509736

HSP90AB1

Heat shock protein 90 kDa alpha (cytosolic), class B member 1

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5 Phosphor ERK (fold) 4 3 2 1 0 + DMSO ABR (1 nM) Inhibitors PD DMSO
(a)

7
Phosphor JNK/SAPK (fold) 5 4 3 2 1 0 +

5 Phosphor P38 (fold)

4 3 2 1 0 +

SB

DMSO

PD ABR PHB 1 Actin 3.2 4.5 12.8 13.2 16.6 Actin 0 3 6 9 12 18 (h) ABR PHB 1 0 3 6

SB 9 12 18 (h) ABR PHB 3.1 4.7 9.8 11.4 15.8 Actin

(b)

SP 0 3 6 9 12 18 (h)

Figure 2: The JNK/SAPK signaling pathway is required for abrin (ABR)-triggered upregulation of prohibitin (PHB). (a) Cells were treated with or without indicated inhibitors for 1 h before ABR treatment. Eects of PD98059 (PD; 20 M), SB203580 (SB; 20 M), or SP600125 (SP; 30 M) on their target signaling molecules were shown. (b) Cells were pretreated with 20 M PD, 20 M SB, or 30 M SP for 1 h before ABR treatment. After the indicated period of time, only SP signicantly inhibited the upregulation of PHB by ABR as shown by western blot analysis.

100 Percentage of apoptosis (%) 80 60 40 20 0 Control siRNA PHB siRNA ABR (1 nM) +

Control siRNA + PHB siRNA ABR (1 nM)

Bax 1 Cleaved-caspase 3 1 +

3.4

0.6

2.7

0.3

Cleaved-PARP

+ + Actin
(b)

9.8

1.7

1 nM ABR 0.1 nM ABR

(a)

Figure 3: Downregulation of prohibitin (PHB) delays abrin (ABR)-triggered cell apoptosis. (a) Downregulation of PHB expression with siRNA delays ABR-triggered cell apoptosis in Jurkat T cells. The cells were treated with 1 nM ABR for 18 h (n = 5). The average SD is shown from separate experiments. P < 0.001. (b) Downregulation of PHB inhibits expression of Bax and activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) 9 h after ABR treatment as shown by western blot analysis.

+ SP 0

+ +

0.4

0.8

5.3

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ABR (1 nM) ABR (1 nM) 0 PHB 1 Tubulin 0.9 0.9 0.8 0.8 0.7 0.4 1 Cytoplasm Actin 1.3 1.4 3.2 Western blot 1 2 3 4 5 6 (h) P53 0 3 6 9 (h)

PHB 1 Sp1
(a)

IP: PHB 1.3 2.3 1.8 1.6 2.1 4.3 Nucleus P53 1 1. 1 1.3
(b)

IB: P53 3.1

IP western

ABR (1 nM)

(h)

PHB Ab ABR (1 nM) 1 IgG 1 Actin 1.3 2.8 4.3 Western blot 1.3 1.5 2.6 ChIP Bax 0 3 6 18 (h)

10% input
(c)

(d)

Figure 4: Prohibitin (PHB) induces the transcriptional activity of p53, which promotes expression of Bax. (a) Abrin (ABR)-induced translocation of PHB from cytoplasm to nucleus. (b) ABR upregulates p53 (western blot) and promotes the interaction between PHB and p53 in cells (immunoprecipitated western blot). (c) Association of PHB with the promoter region of the p53-targeted gene Bax.

that cap-independent protein translation occurs in ABRtriggered apoptosis [42]. Hence, although PHB was rst dened as a mitochondrial protein stabilizer [43], it was later shown to have diverse functions in a variety of processes including senescence, development, and tumor suppression [44]. In addition, PHB enhances the transcriptional activity of the tumor suppressor p53 via physical interaction [29, 45]. Our results are in agreement with earlier ndings that PHB can interact with and upregulate p53 function during apoptosis [29]. It would, therefore, be interesting to determine the involvement of other coactivators in the PHBp53 transcriptional activator complex upon ABR treatment. Indeed, ABR may trigger cell apoptosis through its protein synthesis inhibition, ribotoxic stress, mitochondrial stress, PARP-induced NAD+ depletion, and ROS- and nuclease-induced DNA damage [46]. In addition, others and our previous works showed that ABR-induced apoptosis seems to occur either concomitant with or before the inhibition of protein synthesis [15, 46]. Although we have not yet determined a correlation between the 3 dierent ABR-induced pathways (including depurination activity, AOP-1 interaction, and prohibitin upregulation), both our current study and previous results indicated that either

overexpression of AOP-1 or blockade of PHB expression may signicantly reduce apoptosis (P < 0.05 for each). ABR upregulates the expression of, but does not interact with, PHB. On the contrary, ABR interacts with AOP-1 without upregulating it (data not shown). Although AOP-1 and PHB are thought to share several conserved domains that are expected to play similar roles in normal cells, the reason why they display distinct functions upon ABR treatment is still unclear; this would be an interesting topic for further studies. In addition, it seems that the interaction of ABR with AOP1 is independent of depurination activity, and whether the upregulation of PHB depends on depurination is open to further study. Only an ecient cellular transport system for the toxicity-free mutant (ABR A chain E164Q) would be free of intact protein contamination (e.g., the reassociation of the A chain to the B chain). Moreover, our results here show that early growth response 1 (EGR1), a transcription factor that controls the early growth response and facilitates tissue healing, is signicantly upregulated by ABR in leukemia cells. This result is in agreement with the response of lung epithelial cells to ricin [47], which may well serve as one of the markers of RIP-exerted toxicity. Ongoing studies are evaluating the

Evidence-Based Complementary and Alternative Medicine

ABRA Extracellular ABRB

Cytoplasm

Cellular factors? Translation inhibition

Endosome Golgi Endoplasmic reticulum Nucleus

JNK/SAPK activation

ABRA AOP-1

Mitochondria

PHB gene expression r ROS Lipid peroxidation m Cytochrome C release Caspase 9 Caspase 3 PARP Bax gene expression DNA fragmentation PHB P53 Other factors?

PHB

Apoptosis

Figure 5: A model of abrin (ABR)-triggered apoptosis. ABR-induced apoptosis may occur through at least 3 pathways: rst, inhibition of protein synthesis by its N-glycosidase activity; second, modulation of the function of mitochondria by specic interaction with antioxidant protein-1 (AOP-1); and third, interference with the transcription regulated by prohibitin (PHB). Repression of prohibitin attenuates ABRtriggered apoptosis via preventing the expression of BAX, cleaved-caspase 3, and cleaved-poly(ADP-ribose) polymerase (PARP). Once PHB is upregulated by ABR through the JNK/SAPK signaling pathway, the expression of proapoptotic gene Bax is turned on through the nuclear translocation and p53 interaction of PHB, by which activates the caspase cascade, and nally, apoptosis occurs.

potential of these ABR-related genes for clinical intervention. Nevertheless, as Abrus precatorius is labeled as a biological weapon which may be fatal if eaten, development of a passive vaccine or an antidote for ABR is necessary but under investigation [48, 49]. More understanding of the molecular mechanisms exerted by RIP family proteins may accelerate their clinical applications. In conclusion, we propose the model shown in Figure 5. ABR exhibits biological functions involving at least 3 pathways: translational inhibition, mitochondrial dysfunction, and transcriptional interfere through the upregulation of PHB. Since the downregulation of PHB signicantly delays apoptosis induced by ABR, PHB could be employed in reducing the toxicity of immunotoxins and, hence, improve the eciency of cancer chemotherapy.

References
[1] J. Y. Lin, K. Y. Tserng, C. C. Chen, L. T. Lin, and T. C. Tung, Abrin and ricin: new anti-tumour substances, Nature, vol. 227, no. 5255, pp. 292293, 1970. [2] J. U. Baenziger and D. Fiete, Structural determinants of Ricinus communis agglutinin and toxin specicity for oligosaccharides, Journal of Biological Chemistry, vol. 254, no. 19, pp. 97959799, 1979. [3] F. Stirpe, L. Barbieri, M. G. Battelli, M. Soria, and D. A. Lappi, Ribosome-inactivating proteins from plants: present status and future prospects, Nature Biotechnology, vol. 10, no. 4, pp. 405412, 1992. [4] S. J. Kaufman and A. McPherson, Abrin and hurin: two new lymphocyte mitogens, Cell, vol. 4, no. 3, pp. 263268, 1975. [5] V. P. Kamboj and B. N. Dhawan, Research on plants for fertility regulation in India, Journal of Ethnopharmacology, vol. 6, no. 2, pp. 191226, 1982. [6] J. Y. Lin, T. C. Lee, and T. C. Tung, Inhibitory eects of four isoabrins on the growth of sarcoma 180 cells, Cancer Research, vol. 42, no. 1, pp. 276279, 1982. [7] R. Hegde, T. K. Maiti, and S. K. Podder, Purication and characterization of three toxins and two agglutinins from Abrus precatorius seed by using lactamyl-sepharose anity chromatography, Analytical Biochemistry, vol. 194, no. 1, pp. 101109, 1991.

Acknowledgments
This study was supported by Grants from the Committee on Chinese Medicine and Pharmacy (CCMP-96-RD-203-1) and the Department of Health (DOH97-TD-I-111-TM018, DOH98-TD-I-111-TM020), executive Yuan, Taiwan.

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