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The aberrant expression and localization of prohibitin during apoptosis of human cholangiocarcinoma Mz-ChA-1 cells
FEBS Letters 588 (2014) 422–428
journal homepage: www.FEBSLetters.org
The aberrant expression and localization of prohibitin during apoptosis of human cholangiocarcinoma Mz-ChA-1 cells Wei Song a,b,1, Ling Tian a,1, Shan-Shan Li a, Dong-Yan Shen c,⇑, Qing-Xi Chen a,⇑ a
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361005, China School of Life Sciences and Engineering, Henan University of Urban Construction, Pingdingshan 467044, China c Center Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen 361003, China b
a r t i c l e
i n f o
Article history: Received 25 October 2013 Revised 1 December 2013 Accepted 5 December 2013 Available online 28 December 2013 Edited by Varda Rotter Keywords: Prohibitin Human cholangiocarcinoma Localization shift Interaction
a b s t r a c t In this study, we aimed to investigate the aberrant expression and shift in localization of prohibitin (PHB) during apoptosis of human cholangiocarcinoma cells. Our study demonstrated that PHB was expressed primarily in the cytoplasm and only a little in the nucleus. However, PHB expression significantly decreased, and its localization shifted from the cytoplasm to the nucleus during apoptosis. PHB co-localized with AIF, Rb, p53, and c-Fos, but the region of co-localization was altered after treatment. Meanwhile, we detected a direct interaction between PHB and both p53 and Rb in Mz-ChA-1 cells. These results suggest that the altered localization and expression of PHB, as well as its co-localization with related oncogenes and tumor suppressor genes, can affect the apoptosis of Mz-ChA-1 cells. Structured summary of protein interactions: AIF and PHB colocalize by fluorescence microscopy (View interaction) Rb and PHB colocalize by fluorescence microscopy (View interaction) c-Fos and PHB colocalize by fluorescence microscopy (View interaction) PHB and p53 colocalize by fluorescence microscopy (View interaction) Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Prohibitins (PHB1 and PHB2), referred to here as PHB proteins, are evolutionarily strongly conserved proteins that are present in mitochondria of yeast, plants, and mammals [1–3]. PHB, as also known PHB1, it has been functionally linked to diverse processes, such as transcriptional regulation, cell proliferation, development, and mitochondrial function [4–6]. It was shown that PHB might be closely related with the development of cancer. The expression of PHB was clearly upregulated in gastric cancer, liver cancer, and uterine cancer. Another study demonstrated that over-expression of PHB inhibited proliferation of MCF cells during vitamin D – induced anti-proliferative activity and that PHB promoted apoptosis via interaction with tumor suppressor proteins [7–10]. Therefore, the roles of PHB in carcinogenesis and tumor cell reversal are still unclear.
⇑ Corresponding authors. Fax: +86 592 2185487 (Q.-X. Chen). E-mail addresses: [email protected] (D.-Y. Shen), [email protected] (Q.-X. Chen). 1 These authors contributed equally to this work.
The prohibitins form a high molecular weight complex in the mitochondrial inner membrane, and this complex has been shown to have a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes [2]. PHB is essential for normal mitochondrial development, and PHB deficiency in Caenorhabditis elegans is associated with deficient mitochondrial biogenesis [11]. PHB is also localized to the plasma membrane in certain cell types, and two recent reports suggest that it might function as a surfacebinding site [12,13]. Therefore, the function of PHB depends on its localization in different tumor cell lines. In previous study, we have demonstrated that apoptosis in human cholangiocarcinoma (Mz-ChA-1) [14], human cholangiocarcinoma (QBC-939) [15] and human hepatocarcinoma (SMMC-7721) [16] cells can be induced effectively using the novel inducer ESC3, the active ingredient of crocodile bile. Hereby, based on the inducive effects of ESC-3 on the apoptosis of different human cancer cell lines, we extended our study to examine the aberrant expression of PHB in ESC-3-treated human cholangiocarcinoma Mz-ChA-1 cells. We aimed to investigate the localization shift of PHB in Mz-ChA-1 cells, and ultimately, we explored the relationship of PHB with related oncogenes and tumor suppressor genes. This study aimed to provide additional evidence of the roles of PHB in tumor cell apoptosis.
0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.12.021
W. Song et al. / FEBS Letters 588 (2014) 422–428
2. Materials and methods 2.1. Cell culture and treatment Mz-ChA-1 cells, obtained from China Center for Type Culture Collection, were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C in a humidified atmosphere containing 50 ml/l CO2. Twenty-four hours after seeding, the cells were treated with culture medium containing 20 lg/ml ESC-3. ESC-3 was a mixture of bile acids isolated from crocodile bile using Sephadex LH-20 and RP-18 reversed-phase columns as described in our previous study [14]. 2.2. Two-dimensional gel electrophoresis and image analysis 2-D polyacrylamide gel electrophoresis (PAGE) was performed as follows [17]. Immobilized linear pH 3–10 gradient 18 cm dry strips (Bio-Rad, USA) were used for first-dimension isoelectric focusing. For the second dimension, 12% SDS–polyacrylamide gels were used. The gels were stained using a silver nitrate solution compatible with MS. Image scanning (UMAX Power Look III) and analyses (PD Quest 8.0 software, Bio-Rad) of the three triplicate sets of silver-stained 2D gels were performed. The digitized twodimensional electrophoresis (2-DE) gel images were compared via the matching method. Differentially expressed spots were annotated and analyzed. These spots were cut from the gels. After a series of steps, including silver removal, reduction with DL-dithiothreitol, alkylation with iodoacetamide, and in-gel digestion with trypsin, peptide mass fingerprints (PMFs) were generated using a Bruker III MALDI-TOF mass spectrometer. The spectra were internally calibrated using the trypsin autolysis products [842.51 (M+H) and 2211.11 (M+H)] using Flex Analysis software and were searched against the Swiss-Prot and NCBI databases using the Mascot tool from matrix science. All of the searches were analyzed with a 50 ppm mass tolerance. 2.3. Western blot Western blot analysis was performed as previously described [14]. Briefly, 20 lg cell lysates were loaded and separated on polyacrylamide gels and then transferred onto PVDF membranes (Millipore, Bedford, MA) according to a standard protocol. Non-specific reactivity was blocked by incubating the membranes at 48 °C for 1 h in PBS with 2% BSA. The target proteins were probed using primary antibodies and horseradish peroxidase-labeled secondary antibodies (Santa Cruz). Immunoreactive proteins were detected via an enhanced chemiluminescence (ECL) detection system (Pierce). b-Actin was used as an indicator of equal lane loading.
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at the annealing stage of each cycle. A melting curve was generated during the reactions to measure the occurrence of primer–dimer formation. The 2DDCt method was used to calculate the relative mRNA level of each gene. 2.5. Sample preparation for immunofluorescence microscopy After pre-fixation with 4% paraformaldehyde at room temperature for 10 min, non-specific reactivity was blocked by incubating the membranes at room temperature for 1 h in 3% BSA. The cover slips were incubated with PHB primary antibodies at 37 °C for 1 h. After washing, the cover slips were rinsed in tetramethylrhodamine isothiocyanate (TRITC) fluorescent dye-labeled secondary antibodies for 1 h at room temperature. Each cover slip was mounted using one drop of anti-fluorescence quencher. Afterwards, the cover slips were enveloped using 90% glycerol and observed under a fluorescence microscope. 2.6. Laser scanning confocal microscopic observation of PHB These studies were performed as previously described [18]. MzChA-1 cells and ESC-3-treated cells were seeded on a cover slip and cultured overnight. After washing twice with PBS for 10 min, the cells were immersed in 0.5% Triton X-100 for 5–10 min. After fixation with 4% paraformaldehyde for 10 min, the cells were blocked with 3% BSA for 1 h. The cover slips were incubated with different primary antibody mixtures (PHB/AIF, PHB/c-Fos, PHB/p53, or PHB/ Rb) at 4 °C overnight. After washing, the cover slips were incubated with secondary antibodies (labeled with fluorescein) at 4 °C for 3 h. After sealing with an anti-fluorescence quencher, the cover slip was observed under a laser scanning confocal microscope (TCSSP2 MP). The negative control group was incubated with 3% BSA instead of the primary antibody. 2.7. GST pull-down The samples were incubated with non-carrier bacterial plasmids, and the constructed prokaryotic expressive strains were induced by soluble expression. The GST protein and the GST combined protein were refined using glutathione sepharose 4B following the procedure described below. RIPA lysis buffer was used to dissolve the Mz-ChA-1 cells, which were then centrifuged at 12 000g at 4 °C for 5 min. The centrifuged cell lysates were incubated with GST and GST-PHB combined with beads at 4 °C for 1 h in a silent mixer. Next, they were washed three times using GST washing buffer; 5 SDS loading buffer was added to perform SDS-PAGE, and the same volume was added to the cell lysates as described previously. The interaction between the prey protein and the bait protein was verified by Western blot. 3. Results
2.4. RNA extraction and quantitative real-time PCR 3.1. Proteomic analysis of MZ-CHA-1 cells before and after treatment These studies were performed as previously described [15]. Briefly, total RNA was extracted from cells using the RNAiso Plus Kit (Takara, Japan) following the manufacturer’s instructions. The mRNA level of PHB was determined via quantitative real-time PCR. The primers used for real-time PCR were as follows: 50 -GCAG GACATTGTGGTAGGGG-30 and 50 -GCTGGTGAAGATGCGAGGAA-30 for the forward and reverse primers, respectively. Real-time quantitative PCR was performed using a SYBR PrimeScript RT-PCR Kit in accordance with the manufacturer’s instructions. Then, PCR was using a Rotor-Gene 6000 PCR cycler (Corbett Research, Australia) according to the following protocol: 30 s at 95 °C, one cycle; 10 s at 95 °C and 40 s at 57 °C, 45 cycles. Fluorescence was detected
For proteomic analysis, cell lysates from control and treated cells were subjected to 2D-PAGE. The experiments were independently repeated three times, and representative gel images are shown in Fig. 1A. Scanned gels were quantified using PDQuest 8.0 version (Bio-Rad). Fig. 1B displays the enlarged maps of altered expression of whole cell proteins from Mz-ChA-1 cells. We detected approximately 292 ± 12 proteins spots on the silver-stained gels of the control group, while 310 ± 10 protein spots were detected on the gels of treated group, suggesting that the result of 2-DE PAGE was highly stable and reproducible. Each differentially expressed protein spot was analyzed by MALDI–TOF–MS, and the
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Fig. 1. Two-dimensional protein profiles from Mz-ChA-1 cells. (A) Representative two-dimensional protein profiles from the whole cell proteins of Mz-ChA-1 cells before and after ESC-3 treatment. Proteins were separated on the basis of pI (X-axis) and molecular mass (Y-axis), and visualized by silver staining. The differentially expressed proteins are marked with arrow symbols on the gels. Spots L1–L6 indicate the up-regulated proteins, while spots L7–L13 indicate the down-regulated proteins; L10 indicates the spot of PHB. (B) Enlarged portions from 2-DE gels. (C) Optical density changes in the identified PHB protein spot in the control and treated Mz-ChA-1 cells. Three repeated sets of silver-stained 2D gels were tested for each sample using PD Quest 8.0 software (P < 0.01).
mass fingerprints of the acquired peptides were matched against peptide library data (www.matrixscience.com). The identified proteins are listed in Table 1. The expressions of human ERp44, Ranspecific GTPase-activating protein, proteasome activator complex subunit 1 isoform 1, and human galectin-1 were up-regulated, while the expressions of Rho GDP dissociation inhibitor, tumor protein translationally controlled 1, heterogeneous nuclear ribonucleoprotein K, prohibitin, human heart L-lactate dehydrogenase H chain, leukocyte elastase inhibitor, and S-adenosylhomocysteine hydrolase were down-regulated. Another 31 protein spots were not identified due to their low abundance. The spot encoded L10
was identified as PHB (Fig. 1B; Table 1), and the expression of PHB was found to be reduced in the treated cells (Fig. 1C). 3.2. Confirmation of altered expression of PHB To further verify the aberrant change in PHB expression, Western blot analysis and real-time PCR were employed to confirm the protein and mRNA expression levels, respectively, of PHB in Mz-ChA-1 cells before and after treatment. The Western blot assay results primarily indicated that the level of PHB in the treated cells was much lower than the level of PHB in the control cells, suggesting that the
Table 1 The differential proteins identified by PMF searching against SwissProt Database. Spot No.
Protein name
Up-regulated proteins 1 Chain A, crystal structure of human Erp44 2 Ran-specific GTPase-activating protein 3 Unnamed protein product 4 Proteasome activator complex subunit 1 isoform 1 5 Chain B, X-ray crystal structure of human galectin-1 6 Ran-specific GTPase-activating protein Down-regulated proteins 7 Rho GDP dissociation inhibitor 8 Tumor protein translationally controlled 1 9 Heterogeneous nuclear ribonucleoprotein K 10 Prohibitin 11 Human heart L-lactate Dehydrogenase Chain H 12 Leukocyte elastase inhibitor 13 S-adenosylhomocysteine hydrolase
Accession No.
Protein MW(Da)
Protein pI
Protein score
gi|193885198 gi|4506407 gi|189053448 gi|5453990 gi|42542978 gi|542991
44 284 23 467 48 541 28 876 15 048 23 610
5.04 5.19 5.52 5.78 5.34 5.21
112 130 138 99 136 40
gi|4757768 gi|15214610 gi|55958547 gi|4505773 gi|13786847
23 250 19 759 42 009 29 843 36 769
5.02 4.84 5.43 5.57 5.72
144 66 117 311 122
gi|13489087 gi|178277
42 829 48 254
5.9 6.03
269 216
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expression of PHB after 48 h of treatment was significantly inhibited by ESC-3 (Fig. 2A). Meanwhile, the results of real-time PCR revealed that PHB was also down-regulated after treatment, which is consistent with the change in protein expression (Fig. 2B). 3.3. The localization and expression of PHB in MZ-CHA-1 cells PHB in Mz-ChA-1 cells before and after treatment was labeled with the fluorescent dye TRITC, which fluoresces red under a fluorescence microscope. The nuclei, which were stained with DAPI, were blue in both the control and treated groups. As shown in Fig. 2C, the intensity of red fluorescence of human cholangiocarcinoma Mz-ChA-1 cells was strong and distributed in the cytoplasm and throughout the region of the karyoplasm and around the nucleus. The red fluorescence was stronger in the cytoplasm than in the nucleus. After treatment, the density and distribution of red fluorescence, representing PHB expression, were both altered in the nucleus. The density of red fluorescence was weaker in the cytoplasm and stronger in the nucleus, suggesting that the distribution of PHB was shifted from the cytoplasmic compartment to the nucleus. 3.4. The co-localization and interaction of PHB with AIF, p53, Rb, and c-Fos Mz-ChA-1 cells were double-immunolabeled using primary antibodies for PHB and either AIF, Rb, p53, or c-Fos. The secondary
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antibody against PHB was labeled with TRITC, which emitted red fluorescence; the secondary antibodies against the other proteins were labeled with FITC, which emitted green fluorescence. We employed laser confocal scanning microscopy to detect the changes in co-localization of PHB with related gene products. The regions colocalization appeared as yellow or orange-yellow. In the control cells, the green fluorescence representing AIF was primarily distributed throughout the cell, but was more intense in the cytoplasm than in the nucleus, although the density was not uniform. In the nucleus, only slight scattered green fluorescence was detected. The results of merged fluorescence revealed that PHB co-localized with AIF near the nuclear membrane (Fig. 3A–C). After treatment, the green fluorescence of AIF was greatly enhanced and uniformly distributed in the nucleus. The merged fluorescence revealed that the co-localization of PHB with AIF was clearly present in the nucleus but not in the cytoplasm, suggesting that the co-localization between PHB and AIF disappeared from the cytoplasm adjacent to the nuclear membrane (Fig. 3D–F). In the control cells, the green fluorescence representing p53 was very weak in nucleus and was primarily distributed in the cytoplasm, especially near the nuclear membrane. The yellow merged fluorescence indicated that PHB clearly co-localized with p53 in the nuclear membrane region (Fig. 3G–I). After ESC-3 treatment, the green color was enhanced and was scattered in the nucleus. The merged fluorescence suggested that the region of co-localization between PHB and p53 was clearly in the nucleus
Fig. 2. Expression and localization of PHB in Mz-ChA-1 cells before and after ECS-3 treatment. (A and B) Western blot and RT-PCR assays confirmed the presence and the decreased expression of PHB via ECS-3 treatment. The induction conditions used in this study are consistent with previous study [14]. Actin was used as internal control. (C) Localization of PHB observed under a fluorescence microscope. PHB was labeled with TRITC, which appeared as red under the fluorescence microscope. The nuclei stained by DAPI were blue in both the control and treated groups. The distribution of PHB translocated from the cytoplasm to the nucleus.
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Fig. 3. Cellular co-localization of PHB with several oncogenes and tumor suppressor genes. (A–F) Expression of PHB and AIF in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the co-localization between PHB and AIF. (G–L) Expression of PHB and p53 in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the colocalization between PHB and p53. (M–R) Expression of PHB and Rb in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the co-localization between PHB and Rb. (S–X) Expression of PHB and c-Fos in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the co-localization between PHB and c-Fos.
and was moderately decreased in the cytoplasm near the nuclear membrane (Fig. 3J–L). In the control cells, the FITC-labeled Rb emitting green fluorescence was primarily distributed in the cytoplasm. The density of the fluorescence at the periphery of the nucleus was relatively stronger, while the fluorescence within the nucleus was weaker. The merged yellow fluorescence revealed that PHB co-localized with Rb in the cytoplasmic compartment, especially near the nucleus (Fig. 3M–O). After ESC-3 treatment, the green Rb fluorescence was enhanced and primarily distributed in the nucleus but had weakened in the cytoplasm. The orange fluorescence appeared in the nucleus, suggesting that the co-localization of PHB with Rb shifted from the cytoplasm to the nucleus (Fig. 3P–R). In the control cells, according to the results of the fluorescence staining, the green fluorescence representing c-Fos expression was well-distributed throughout the cytoplasm. In the nucleus, only slight scattered green fluorescence was observed. The result of
merged fluorescence revealed that PHB co-localized with c-Fos in the cytoplasm, especially near the nuclear membrane (Fig. 3S–U). After treatment with ESC-3, the green c-Fos fluorescence was greatly decreased in the cytoplasm and was primarily localized to the nucleus. The merged yellow fluorescence suggested that the co-localization of PHB with c-Fos decreased in the cytoplasm and increased in the nucleus, indicating that the co-localization of PHB with c-Fos shifted from the cytoplasm to the nucleus (Fig. 3V–X). 3.5. The interaction between PHB and AIF, p53, Rb, and c-Fos in vitro To further verify the interaction between PHB and these oncogene proteins in vitro, a GST pull-down assay was employed. Both GST-PHB and GST were expressed in bacterial strain BL21 and were purified to near homogeneity via GST affinity chromatography (Fig. 4A and B). As shown in Fig. 4C, after the recombinant proteins
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of GST-PHB and GST were incubated with the cell lysate supernatant, the Western blot results indicated that direct interactions existed between GST-PHB and p53 in vitro, but no interaction existed between GST and p53. Identical results appeared between GSTPHB and Rb, but no significant interaction was detected between GST-PHB and either AIF or c-Fos (data not shown). According to these results, we inferred that there is a direct interaction between PHB and both p53 and Rb, which was consistent with the co-localization results in Mz-ChA-1 cells. 4. Discussion The alteration of cellular components is closely related to cell carcinogenesis [19,20]. To further investigate the effects of apoptosis inducer ESC-3 on the protein expression of human cholangiocarcinoma cancer cells, proteomic analysis was employed to screen for the aberrant proteins of human cholangiocarcinoma cells before and after treatment. Our study demonstrated the existence and differential expression of PHB in Mz-ChA-1 cells after ESC-3 treatment. The expression of PHB was dramatically down-regulated in ESC-3 treated cells. Changes in the protein expression level were confirmed via Western blot and RT-PCR assays. Despite the evidence that PHB has anti-tumorigenic properties, there are also a number of studies indicating a pro-tumorigenic role of PHB. The role of PHB in cancer cell proliferation and/or tumor suppression remains controversial [21]. Our results showed that PHB was down-regulated during apoptosis of Mz-ChA-1 cells, which suggested that PHB may be pro-tumorigenic rather than a tumor suppressor. Here, we postulated that PHB might bidirectionally regulate proliferation and apoptosis in different cell lines and apoptosis stages. The change in PHB expression and
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subcellular localization suggested that prohibitin might be a target protein of ESC-3 which participated in the regulation of apoptosis of Mz-ChA-1 cells. PHB was primarily expressed in the cytoplasm but only a little in the nucleus. A clear translocation from the cytoplasm to the nucleus was observed. Our data primarily illustrated the subcellular distribution of PHB. These results suggest that the alteration of expression and subcellular distribution are closely related to the process of apoptosis in Mz-ChA-1 cells. As an important differentially expressed protein, PHB might play pivotal roles in the regulation of human cholangiocarcinoma Mz-ChA-1 cell apoptosis. The involvement of PHB in essential biological processes, such as metabolism, cell proliferation, and development, implicates a critical pleiotropic role for PHB. In this study, we revealed that there was co-localization between PHB and c-Fos, Rb, p53, and AIF in Mz-ChA-1 cells that were altered with treatment, as measured by immunofluorescence microscopy and laser scanning confocal microscopy. During apoptosis, AIF is released from the mitochondrial intermembrane space to the cytosol and the nucleus [22]. PHB plays a critical role in maintaining normal mitochondrial function and morphology [21]. Therefore, we postulated that a close relationship could occur between PHB and AIF during apoptosis of MzChA-1 cells. In our study, the co-localization between PHB and AIF disappeared from the cytoplasm near the nuclear membrane after treatment. For the first time, our study presented the co-localization of PHB with AIF, suggesting PHB mediates the apoptosis of cholangiocarcinoma Mz-ChA-1 cells via AIF release from mitochondria to the nucleus. PHB has also been shown to interact with the retinoblastoma tumor suppressor protein (Rb) and its family members, p107 and p130, in the nucleus [23]. The Rb family suppresses growth via
Fig. 4. The interactions between PHB and both p53 and Rb in vitro. (A) Expression of GST-PHB in E. coli strain BL21 induced by IPTG. Ovals indicate GST-PHB protein bands. (B) Purification of PHB using GST affinity chromatography (Amersham Biosciences) followed by size exclusion chromatography using a Superdex 200 column (Amersham Biosciences). (C) GST pull-down assay was employed to verify the interaction between PHB and both p53 and Rb in MZ-CHA-1 cells in vitro. Specifically bound proteins were resolved via SDS–polyacrylamide gel electrophoresis and detected via Western blot.
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binding to and inhibiting the E2F family of transcription factors [24]. PHB was shown to repress E2F-mediated transcription via its interaction with Rb and histone deacetylase [25]. In our study, we found that the region of co-localization between PHB and p53 was clearly in the nucleus and moderately decreased in the cytoplasm near the nuclear membrane after treatment with ESC-3. Obviously, this alteration is correlated with the proliferation and apoptosis of Mz-ChA-1 cells because the region of the nuclear lamina is an active site of DNA replication and transcription; this synergistic translocation of PHB and p53 may influence the expression of p53 and its downstream genes in Mz-ChA-1 cells, resulting in tumor cell reversion. Similarly, co-localization between PHB and Rb was present in the cytoplasmic region, especially near the nucleus. However, we also observed that the co-localization of PHB and Rb translocated from the cytoplasm to the nucleus after treatment. The interaction between PHB and both p53 and Rb were further verified via the GST pull-down experiment. This phenomenon illustrates that the alteration of PHB trafficking and its enhanced co-localization with Rb in the nucleus may repress the ability of E2F to activate the expression of its downstream genes, subsequently controlling cell proliferation and promoting apoptosis. Previous studies showed that PHB and c-Fos were regulated by Rb, and the expression of PHB and c-Fos was increased in cells in which Rb was over-expressed [26,27]. The co-localization between PHB and Rb was mentioned above; we observed that there was distinct co-localization between PHB and c-Fos in the nucleus. These results bring are consistent with the altered region of co-localization between PHB and Rb, indicating that c-Fos and Rb preceded the process of regulation of apoptosis in Mz-ChA-1 cells after ESC-3 treatment. However, these results were not verified in our GST pull-down experiment. Therefore, further research on the cFos protein is needed, as we could not find published research on the interaction between PHB and c-Fos in apoptotic cells. Our study primarily analyzed and revealed the aberrant location and expression of PHB in ESC-3-induced apoptosis of MzChA-1 cells, as well as the interaction of PHB with oncogenes and tumor suppressor genes. Our findings will help to elucidate the function of PHB during apoptosis of Mz-ChA-1 cells, and the relationships between PHB and tumor related genes have offered new insight into the mechanism of apoptosis of human cholangiocarcinoma cells. Acknowledgments The present investigation was supported by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant No. J1310027/J0106) and by the Science and Technology Foundation of Xiamen, China (No. 3502Z20133009) and by National Nature Science Foundation of China (No. 81101502). References [1] Snedden, W.A. and Fromm, H. (1997) Characterization of the plant homologue of prohibitin, a gene associated with antiproliferative activity in mammalian cells. Plant Mol. Biol. 33, 753–756. [2] Nijtmans, L.G. et al. (2000) Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins. EMBO J. 19, 2444–2451. [3] Coates, P.J., Jamieson, D.J., Smart, K., Prescott, A.R. and Hall, P.A. (1997) The prohibitin family of mitochondrial proteins regulate replicative lifespan. Curr. Biol. 7, 607–610.
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[21] Theiss, A.L. and Sitaraman, S.V. (2011) The role and therapeutic potential of prohibitin in disease. Biochim. Biophys. Acta 1813, 1137–1143. [22] Scovassi, A.I., Soldani, C., Veneroni, P., Bottone, M.G. and Pellicciari, C. (2009) Changes of mitochondria and relocation of the apoptosis inducing factor during apoptosis. Ann. N. Y. Acad. Sci. 1171, 12–17. [23] Wang, S., Nath, N., Adlam, M. and Chellappan, S. (1999) Prohibitin, a potential tumor suppressor, interacts with RB and regulates E2F function. Oncogene 18, 3501. [24] Andrusiak, M.G., McClellan, K.A., Dugal-Tessier, D., Julian, L.M., Rodrigues, S.P., Park, D.S., Kennedy, T.E. and Slack, R.S. (2011) Rb/E2F regulates expression of neogenin during neuronal migration. Mol. Cell Biol. 31, 238–247. [25] Wang, S., Fusaro, G., Padmanabhan, J. and Chellappan, S.P. (2002) Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression. Oncogene 21, 8388. [26] Ito, K. et al. 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The aberrant expression and localization of prohibitin during apoptosis of human cholangiocarcinoma Mz-ChA-1 cells Wei Song a,b,1, Ling Tian a,1, Shan-Shan Li a, Dong-Yan Shen c,⇑, Qing-Xi Chen a,⇑ a
State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen 361005, China School of Life Sciences and Engineering, Henan University of Urban Construction, Pingdingshan 467044, China c Center Laboratory, The First Affiliated Hospital of Xiamen University, Xiamen 361003, China b
a r t i c l e
i n f o
Article history: Received 25 October 2013 Revised 1 December 2013 Accepted 5 December 2013 Available online 28 December 2013 Edited by Varda Rotter Keywords: Prohibitin Human cholangiocarcinoma Localization shift Interaction
a b s t r a c t In this study, we aimed to investigate the aberrant expression and shift in localization of prohibitin (PHB) during apoptosis of human cholangiocarcinoma cells. Our study demonstrated that PHB was expressed primarily in the cytoplasm and only a little in the nucleus. However, PHB expression significantly decreased, and its localization shifted from the cytoplasm to the nucleus during apoptosis. PHB co-localized with AIF, Rb, p53, and c-Fos, but the region of co-localization was altered after treatment. Meanwhile, we detected a direct interaction between PHB and both p53 and Rb in Mz-ChA-1 cells. These results suggest that the altered localization and expression of PHB, as well as its co-localization with related oncogenes and tumor suppressor genes, can affect the apoptosis of Mz-ChA-1 cells. Structured summary of protein interactions: AIF and PHB colocalize by fluorescence microscopy (View interaction) Rb and PHB colocalize by fluorescence microscopy (View interaction) c-Fos and PHB colocalize by fluorescence microscopy (View interaction) PHB and p53 colocalize by fluorescence microscopy (View interaction) Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Prohibitins (PHB1 and PHB2), referred to here as PHB proteins, are evolutionarily strongly conserved proteins that are present in mitochondria of yeast, plants, and mammals [1–3]. PHB, as also known PHB1, it has been functionally linked to diverse processes, such as transcriptional regulation, cell proliferation, development, and mitochondrial function [4–6]. It was shown that PHB might be closely related with the development of cancer. The expression of PHB was clearly upregulated in gastric cancer, liver cancer, and uterine cancer. Another study demonstrated that over-expression of PHB inhibited proliferation of MCF cells during vitamin D – induced anti-proliferative activity and that PHB promoted apoptosis via interaction with tumor suppressor proteins [7–10]. Therefore, the roles of PHB in carcinogenesis and tumor cell reversal are still unclear.
⇑ Corresponding authors. Fax: +86 592 2185487 (Q.-X. Chen). E-mail addresses: [email protected] (D.-Y. Shen), [email protected] (Q.-X. Chen). 1 These authors contributed equally to this work.
The prohibitins form a high molecular weight complex in the mitochondrial inner membrane, and this complex has been shown to have a role in the stabilization of newly synthesized subunits of mitochondrial respiratory enzymes [2]. PHB is essential for normal mitochondrial development, and PHB deficiency in Caenorhabditis elegans is associated with deficient mitochondrial biogenesis [11]. PHB is also localized to the plasma membrane in certain cell types, and two recent reports suggest that it might function as a surfacebinding site [12,13]. Therefore, the function of PHB depends on its localization in different tumor cell lines. In previous study, we have demonstrated that apoptosis in human cholangiocarcinoma (Mz-ChA-1) [14], human cholangiocarcinoma (QBC-939) [15] and human hepatocarcinoma (SMMC-7721) [16] cells can be induced effectively using the novel inducer ESC3, the active ingredient of crocodile bile. Hereby, based on the inducive effects of ESC-3 on the apoptosis of different human cancer cell lines, we extended our study to examine the aberrant expression of PHB in ESC-3-treated human cholangiocarcinoma Mz-ChA-1 cells. We aimed to investigate the localization shift of PHB in Mz-ChA-1 cells, and ultimately, we explored the relationship of PHB with related oncogenes and tumor suppressor genes. This study aimed to provide additional evidence of the roles of PHB in tumor cell apoptosis.
0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.12.021
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2. Materials and methods 2.1. Cell culture and treatment Mz-ChA-1 cells, obtained from China Center for Type Culture Collection, were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C in a humidified atmosphere containing 50 ml/l CO2. Twenty-four hours after seeding, the cells were treated with culture medium containing 20 lg/ml ESC-3. ESC-3 was a mixture of bile acids isolated from crocodile bile using Sephadex LH-20 and RP-18 reversed-phase columns as described in our previous study [14]. 2.2. Two-dimensional gel electrophoresis and image analysis 2-D polyacrylamide gel electrophoresis (PAGE) was performed as follows [17]. Immobilized linear pH 3–10 gradient 18 cm dry strips (Bio-Rad, USA) were used for first-dimension isoelectric focusing. For the second dimension, 12% SDS–polyacrylamide gels were used. The gels were stained using a silver nitrate solution compatible with MS. Image scanning (UMAX Power Look III) and analyses (PD Quest 8.0 software, Bio-Rad) of the three triplicate sets of silver-stained 2D gels were performed. The digitized twodimensional electrophoresis (2-DE) gel images were compared via the matching method. Differentially expressed spots were annotated and analyzed. These spots were cut from the gels. After a series of steps, including silver removal, reduction with DL-dithiothreitol, alkylation with iodoacetamide, and in-gel digestion with trypsin, peptide mass fingerprints (PMFs) were generated using a Bruker III MALDI-TOF mass spectrometer. The spectra were internally calibrated using the trypsin autolysis products [842.51 (M+H) and 2211.11 (M+H)] using Flex Analysis software and were searched against the Swiss-Prot and NCBI databases using the Mascot tool from matrix science. All of the searches were analyzed with a 50 ppm mass tolerance. 2.3. Western blot Western blot analysis was performed as previously described [14]. Briefly, 20 lg cell lysates were loaded and separated on polyacrylamide gels and then transferred onto PVDF membranes (Millipore, Bedford, MA) according to a standard protocol. Non-specific reactivity was blocked by incubating the membranes at 48 °C for 1 h in PBS with 2% BSA. The target proteins were probed using primary antibodies and horseradish peroxidase-labeled secondary antibodies (Santa Cruz). Immunoreactive proteins were detected via an enhanced chemiluminescence (ECL) detection system (Pierce). b-Actin was used as an indicator of equal lane loading.
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at the annealing stage of each cycle. A melting curve was generated during the reactions to measure the occurrence of primer–dimer formation. The 2DDCt method was used to calculate the relative mRNA level of each gene. 2.5. Sample preparation for immunofluorescence microscopy After pre-fixation with 4% paraformaldehyde at room temperature for 10 min, non-specific reactivity was blocked by incubating the membranes at room temperature for 1 h in 3% BSA. The cover slips were incubated with PHB primary antibodies at 37 °C for 1 h. After washing, the cover slips were rinsed in tetramethylrhodamine isothiocyanate (TRITC) fluorescent dye-labeled secondary antibodies for 1 h at room temperature. Each cover slip was mounted using one drop of anti-fluorescence quencher. Afterwards, the cover slips were enveloped using 90% glycerol and observed under a fluorescence microscope. 2.6. Laser scanning confocal microscopic observation of PHB These studies were performed as previously described [18]. MzChA-1 cells and ESC-3-treated cells were seeded on a cover slip and cultured overnight. After washing twice with PBS for 10 min, the cells were immersed in 0.5% Triton X-100 for 5–10 min. After fixation with 4% paraformaldehyde for 10 min, the cells were blocked with 3% BSA for 1 h. The cover slips were incubated with different primary antibody mixtures (PHB/AIF, PHB/c-Fos, PHB/p53, or PHB/ Rb) at 4 °C overnight. After washing, the cover slips were incubated with secondary antibodies (labeled with fluorescein) at 4 °C for 3 h. After sealing with an anti-fluorescence quencher, the cover slip was observed under a laser scanning confocal microscope (TCSSP2 MP). The negative control group was incubated with 3% BSA instead of the primary antibody. 2.7. GST pull-down The samples were incubated with non-carrier bacterial plasmids, and the constructed prokaryotic expressive strains were induced by soluble expression. The GST protein and the GST combined protein were refined using glutathione sepharose 4B following the procedure described below. RIPA lysis buffer was used to dissolve the Mz-ChA-1 cells, which were then centrifuged at 12 000g at 4 °C for 5 min. The centrifuged cell lysates were incubated with GST and GST-PHB combined with beads at 4 °C for 1 h in a silent mixer. Next, they were washed three times using GST washing buffer; 5 SDS loading buffer was added to perform SDS-PAGE, and the same volume was added to the cell lysates as described previously. The interaction between the prey protein and the bait protein was verified by Western blot. 3. Results
2.4. RNA extraction and quantitative real-time PCR 3.1. Proteomic analysis of MZ-CHA-1 cells before and after treatment These studies were performed as previously described [15]. Briefly, total RNA was extracted from cells using the RNAiso Plus Kit (Takara, Japan) following the manufacturer’s instructions. The mRNA level of PHB was determined via quantitative real-time PCR. The primers used for real-time PCR were as follows: 50 -GCAG GACATTGTGGTAGGGG-30 and 50 -GCTGGTGAAGATGCGAGGAA-30 for the forward and reverse primers, respectively. Real-time quantitative PCR was performed using a SYBR PrimeScript RT-PCR Kit in accordance with the manufacturer’s instructions. Then, PCR was using a Rotor-Gene 6000 PCR cycler (Corbett Research, Australia) according to the following protocol: 30 s at 95 °C, one cycle; 10 s at 95 °C and 40 s at 57 °C, 45 cycles. Fluorescence was detected
For proteomic analysis, cell lysates from control and treated cells were subjected to 2D-PAGE. The experiments were independently repeated three times, and representative gel images are shown in Fig. 1A. Scanned gels were quantified using PDQuest 8.0 version (Bio-Rad). Fig. 1B displays the enlarged maps of altered expression of whole cell proteins from Mz-ChA-1 cells. We detected approximately 292 ± 12 proteins spots on the silver-stained gels of the control group, while 310 ± 10 protein spots were detected on the gels of treated group, suggesting that the result of 2-DE PAGE was highly stable and reproducible. Each differentially expressed protein spot was analyzed by MALDI–TOF–MS, and the
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Fig. 1. Two-dimensional protein profiles from Mz-ChA-1 cells. (A) Representative two-dimensional protein profiles from the whole cell proteins of Mz-ChA-1 cells before and after ESC-3 treatment. Proteins were separated on the basis of pI (X-axis) and molecular mass (Y-axis), and visualized by silver staining. The differentially expressed proteins are marked with arrow symbols on the gels. Spots L1–L6 indicate the up-regulated proteins, while spots L7–L13 indicate the down-regulated proteins; L10 indicates the spot of PHB. (B) Enlarged portions from 2-DE gels. (C) Optical density changes in the identified PHB protein spot in the control and treated Mz-ChA-1 cells. Three repeated sets of silver-stained 2D gels were tested for each sample using PD Quest 8.0 software (P < 0.01).
mass fingerprints of the acquired peptides were matched against peptide library data (www.matrixscience.com). The identified proteins are listed in Table 1. The expressions of human ERp44, Ranspecific GTPase-activating protein, proteasome activator complex subunit 1 isoform 1, and human galectin-1 were up-regulated, while the expressions of Rho GDP dissociation inhibitor, tumor protein translationally controlled 1, heterogeneous nuclear ribonucleoprotein K, prohibitin, human heart L-lactate dehydrogenase H chain, leukocyte elastase inhibitor, and S-adenosylhomocysteine hydrolase were down-regulated. Another 31 protein spots were not identified due to their low abundance. The spot encoded L10
was identified as PHB (Fig. 1B; Table 1), and the expression of PHB was found to be reduced in the treated cells (Fig. 1C). 3.2. Confirmation of altered expression of PHB To further verify the aberrant change in PHB expression, Western blot analysis and real-time PCR were employed to confirm the protein and mRNA expression levels, respectively, of PHB in Mz-ChA-1 cells before and after treatment. The Western blot assay results primarily indicated that the level of PHB in the treated cells was much lower than the level of PHB in the control cells, suggesting that the
Table 1 The differential proteins identified by PMF searching against SwissProt Database. Spot No.
Protein name
Up-regulated proteins 1 Chain A, crystal structure of human Erp44 2 Ran-specific GTPase-activating protein 3 Unnamed protein product 4 Proteasome activator complex subunit 1 isoform 1 5 Chain B, X-ray crystal structure of human galectin-1 6 Ran-specific GTPase-activating protein Down-regulated proteins 7 Rho GDP dissociation inhibitor 8 Tumor protein translationally controlled 1 9 Heterogeneous nuclear ribonucleoprotein K 10 Prohibitin 11 Human heart L-lactate Dehydrogenase Chain H 12 Leukocyte elastase inhibitor 13 S-adenosylhomocysteine hydrolase
Accession No.
Protein MW(Da)
Protein pI
Protein score
gi|193885198 gi|4506407 gi|189053448 gi|5453990 gi|42542978 gi|542991
44 284 23 467 48 541 28 876 15 048 23 610
5.04 5.19 5.52 5.78 5.34 5.21
112 130 138 99 136 40
gi|4757768 gi|15214610 gi|55958547 gi|4505773 gi|13786847
23 250 19 759 42 009 29 843 36 769
5.02 4.84 5.43 5.57 5.72
144 66 117 311 122
gi|13489087 gi|178277
42 829 48 254
5.9 6.03
269 216
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expression of PHB after 48 h of treatment was significantly inhibited by ESC-3 (Fig. 2A). Meanwhile, the results of real-time PCR revealed that PHB was also down-regulated after treatment, which is consistent with the change in protein expression (Fig. 2B). 3.3. The localization and expression of PHB in MZ-CHA-1 cells PHB in Mz-ChA-1 cells before and after treatment was labeled with the fluorescent dye TRITC, which fluoresces red under a fluorescence microscope. The nuclei, which were stained with DAPI, were blue in both the control and treated groups. As shown in Fig. 2C, the intensity of red fluorescence of human cholangiocarcinoma Mz-ChA-1 cells was strong and distributed in the cytoplasm and throughout the region of the karyoplasm and around the nucleus. The red fluorescence was stronger in the cytoplasm than in the nucleus. After treatment, the density and distribution of red fluorescence, representing PHB expression, were both altered in the nucleus. The density of red fluorescence was weaker in the cytoplasm and stronger in the nucleus, suggesting that the distribution of PHB was shifted from the cytoplasmic compartment to the nucleus. 3.4. The co-localization and interaction of PHB with AIF, p53, Rb, and c-Fos Mz-ChA-1 cells were double-immunolabeled using primary antibodies for PHB and either AIF, Rb, p53, or c-Fos. The secondary
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antibody against PHB was labeled with TRITC, which emitted red fluorescence; the secondary antibodies against the other proteins were labeled with FITC, which emitted green fluorescence. We employed laser confocal scanning microscopy to detect the changes in co-localization of PHB with related gene products. The regions colocalization appeared as yellow or orange-yellow. In the control cells, the green fluorescence representing AIF was primarily distributed throughout the cell, but was more intense in the cytoplasm than in the nucleus, although the density was not uniform. In the nucleus, only slight scattered green fluorescence was detected. The results of merged fluorescence revealed that PHB co-localized with AIF near the nuclear membrane (Fig. 3A–C). After treatment, the green fluorescence of AIF was greatly enhanced and uniformly distributed in the nucleus. The merged fluorescence revealed that the co-localization of PHB with AIF was clearly present in the nucleus but not in the cytoplasm, suggesting that the co-localization between PHB and AIF disappeared from the cytoplasm adjacent to the nuclear membrane (Fig. 3D–F). In the control cells, the green fluorescence representing p53 was very weak in nucleus and was primarily distributed in the cytoplasm, especially near the nuclear membrane. The yellow merged fluorescence indicated that PHB clearly co-localized with p53 in the nuclear membrane region (Fig. 3G–I). After ESC-3 treatment, the green color was enhanced and was scattered in the nucleus. The merged fluorescence suggested that the region of co-localization between PHB and p53 was clearly in the nucleus
Fig. 2. Expression and localization of PHB in Mz-ChA-1 cells before and after ECS-3 treatment. (A and B) Western blot and RT-PCR assays confirmed the presence and the decreased expression of PHB via ECS-3 treatment. The induction conditions used in this study are consistent with previous study [14]. Actin was used as internal control. (C) Localization of PHB observed under a fluorescence microscope. PHB was labeled with TRITC, which appeared as red under the fluorescence microscope. The nuclei stained by DAPI were blue in both the control and treated groups. The distribution of PHB translocated from the cytoplasm to the nucleus.
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Fig. 3. Cellular co-localization of PHB with several oncogenes and tumor suppressor genes. (A–F) Expression of PHB and AIF in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the co-localization between PHB and AIF. (G–L) Expression of PHB and p53 in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the colocalization between PHB and p53. (M–R) Expression of PHB and Rb in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the co-localization between PHB and Rb. (S–X) Expression of PHB and c-Fos in MZ-CHA-1 cells. The yellow fluorescence in the overlay indicates the co-localization between PHB and c-Fos.
and was moderately decreased in the cytoplasm near the nuclear membrane (Fig. 3J–L). In the control cells, the FITC-labeled Rb emitting green fluorescence was primarily distributed in the cytoplasm. The density of the fluorescence at the periphery of the nucleus was relatively stronger, while the fluorescence within the nucleus was weaker. The merged yellow fluorescence revealed that PHB co-localized with Rb in the cytoplasmic compartment, especially near the nucleus (Fig. 3M–O). After ESC-3 treatment, the green Rb fluorescence was enhanced and primarily distributed in the nucleus but had weakened in the cytoplasm. The orange fluorescence appeared in the nucleus, suggesting that the co-localization of PHB with Rb shifted from the cytoplasm to the nucleus (Fig. 3P–R). In the control cells, according to the results of the fluorescence staining, the green fluorescence representing c-Fos expression was well-distributed throughout the cytoplasm. In the nucleus, only slight scattered green fluorescence was observed. The result of
merged fluorescence revealed that PHB co-localized with c-Fos in the cytoplasm, especially near the nuclear membrane (Fig. 3S–U). After treatment with ESC-3, the green c-Fos fluorescence was greatly decreased in the cytoplasm and was primarily localized to the nucleus. The merged yellow fluorescence suggested that the co-localization of PHB with c-Fos decreased in the cytoplasm and increased in the nucleus, indicating that the co-localization of PHB with c-Fos shifted from the cytoplasm to the nucleus (Fig. 3V–X). 3.5. The interaction between PHB and AIF, p53, Rb, and c-Fos in vitro To further verify the interaction between PHB and these oncogene proteins in vitro, a GST pull-down assay was employed. Both GST-PHB and GST were expressed in bacterial strain BL21 and were purified to near homogeneity via GST affinity chromatography (Fig. 4A and B). As shown in Fig. 4C, after the recombinant proteins
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of GST-PHB and GST were incubated with the cell lysate supernatant, the Western blot results indicated that direct interactions existed between GST-PHB and p53 in vitro, but no interaction existed between GST and p53. Identical results appeared between GSTPHB and Rb, but no significant interaction was detected between GST-PHB and either AIF or c-Fos (data not shown). According to these results, we inferred that there is a direct interaction between PHB and both p53 and Rb, which was consistent with the co-localization results in Mz-ChA-1 cells. 4. Discussion The alteration of cellular components is closely related to cell carcinogenesis [19,20]. To further investigate the effects of apoptosis inducer ESC-3 on the protein expression of human cholangiocarcinoma cancer cells, proteomic analysis was employed to screen for the aberrant proteins of human cholangiocarcinoma cells before and after treatment. Our study demonstrated the existence and differential expression of PHB in Mz-ChA-1 cells after ESC-3 treatment. The expression of PHB was dramatically down-regulated in ESC-3 treated cells. Changes in the protein expression level were confirmed via Western blot and RT-PCR assays. Despite the evidence that PHB has anti-tumorigenic properties, there are also a number of studies indicating a pro-tumorigenic role of PHB. The role of PHB in cancer cell proliferation and/or tumor suppression remains controversial [21]. Our results showed that PHB was down-regulated during apoptosis of Mz-ChA-1 cells, which suggested that PHB may be pro-tumorigenic rather than a tumor suppressor. Here, we postulated that PHB might bidirectionally regulate proliferation and apoptosis in different cell lines and apoptosis stages. The change in PHB expression and
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subcellular localization suggested that prohibitin might be a target protein of ESC-3 which participated in the regulation of apoptosis of Mz-ChA-1 cells. PHB was primarily expressed in the cytoplasm but only a little in the nucleus. A clear translocation from the cytoplasm to the nucleus was observed. Our data primarily illustrated the subcellular distribution of PHB. These results suggest that the alteration of expression and subcellular distribution are closely related to the process of apoptosis in Mz-ChA-1 cells. As an important differentially expressed protein, PHB might play pivotal roles in the regulation of human cholangiocarcinoma Mz-ChA-1 cell apoptosis. The involvement of PHB in essential biological processes, such as metabolism, cell proliferation, and development, implicates a critical pleiotropic role for PHB. In this study, we revealed that there was co-localization between PHB and c-Fos, Rb, p53, and AIF in Mz-ChA-1 cells that were altered with treatment, as measured by immunofluorescence microscopy and laser scanning confocal microscopy. During apoptosis, AIF is released from the mitochondrial intermembrane space to the cytosol and the nucleus [22]. PHB plays a critical role in maintaining normal mitochondrial function and morphology [21]. Therefore, we postulated that a close relationship could occur between PHB and AIF during apoptosis of MzChA-1 cells. In our study, the co-localization between PHB and AIF disappeared from the cytoplasm near the nuclear membrane after treatment. For the first time, our study presented the co-localization of PHB with AIF, suggesting PHB mediates the apoptosis of cholangiocarcinoma Mz-ChA-1 cells via AIF release from mitochondria to the nucleus. PHB has also been shown to interact with the retinoblastoma tumor suppressor protein (Rb) and its family members, p107 and p130, in the nucleus [23]. The Rb family suppresses growth via
Fig. 4. The interactions between PHB and both p53 and Rb in vitro. (A) Expression of GST-PHB in E. coli strain BL21 induced by IPTG. Ovals indicate GST-PHB protein bands. (B) Purification of PHB using GST affinity chromatography (Amersham Biosciences) followed by size exclusion chromatography using a Superdex 200 column (Amersham Biosciences). (C) GST pull-down assay was employed to verify the interaction between PHB and both p53 and Rb in MZ-CHA-1 cells in vitro. Specifically bound proteins were resolved via SDS–polyacrylamide gel electrophoresis and detected via Western blot.
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binding to and inhibiting the E2F family of transcription factors [24]. PHB was shown to repress E2F-mediated transcription via its interaction with Rb and histone deacetylase [25]. In our study, we found that the region of co-localization between PHB and p53 was clearly in the nucleus and moderately decreased in the cytoplasm near the nuclear membrane after treatment with ESC-3. Obviously, this alteration is correlated with the proliferation and apoptosis of Mz-ChA-1 cells because the region of the nuclear lamina is an active site of DNA replication and transcription; this synergistic translocation of PHB and p53 may influence the expression of p53 and its downstream genes in Mz-ChA-1 cells, resulting in tumor cell reversion. Similarly, co-localization between PHB and Rb was present in the cytoplasmic region, especially near the nucleus. However, we also observed that the co-localization of PHB and Rb translocated from the cytoplasm to the nucleus after treatment. The interaction between PHB and both p53 and Rb were further verified via the GST pull-down experiment. This phenomenon illustrates that the alteration of PHB trafficking and its enhanced co-localization with Rb in the nucleus may repress the ability of E2F to activate the expression of its downstream genes, subsequently controlling cell proliferation and promoting apoptosis. Previous studies showed that PHB and c-Fos were regulated by Rb, and the expression of PHB and c-Fos was increased in cells in which Rb was over-expressed [26,27]. The co-localization between PHB and Rb was mentioned above; we observed that there was distinct co-localization between PHB and c-Fos in the nucleus. These results bring are consistent with the altered region of co-localization between PHB and Rb, indicating that c-Fos and Rb preceded the process of regulation of apoptosis in Mz-ChA-1 cells after ESC-3 treatment. However, these results were not verified in our GST pull-down experiment. Therefore, further research on the cFos protein is needed, as we could not find published research on the interaction between PHB and c-Fos in apoptotic cells. Our study primarily analyzed and revealed the aberrant location and expression of PHB in ESC-3-induced apoptosis of MzChA-1 cells, as well as the interaction of PHB with oncogenes and tumor suppressor genes. Our findings will help to elucidate the function of PHB during apoptosis of Mz-ChA-1 cells, and the relationships between PHB and tumor related genes have offered new insight into the mechanism of apoptosis of human cholangiocarcinoma cells. Acknowledgments The present investigation was supported by the National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China (Grant No. J1310027/J0106) and by the Science and Technology Foundation of Xiamen, China (No. 3502Z20133009) and by National Nature Science Foundation of China (No. 81101502). References [1] Snedden, W.A. and Fromm, H. (1997) Characterization of the plant homologue of prohibitin, a gene associated with antiproliferative activity in mammalian cells. Plant Mol. Biol. 33, 753–756. [2] Nijtmans, L.G. et al. (2000) Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins. EMBO J. 19, 2444–2451. [3] Coates, P.J., Jamieson, D.J., Smart, K., Prescott, A.R. and Hall, P.A. (1997) The prohibitin family of mitochondrial proteins regulate replicative lifespan. Curr. Biol. 7, 607–610.
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