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Potentiation of Apoptin-Induced Apoptosis by Cecropin B-Like Antibacterial Peptide ABPs1 in Human HeLa Cervical Cancer Cell Lines is Associated with Membrane Pore Formation and Caspase-3 Activation
Potentiation of Apoptin-Induced Apoptosis by Cecropin B-Like Antibacterial Peptide ABPs1 in Human HeLa Cervical Cancer Cell Lines is Associated with Membrane Pore Formation and Caspase-3 Activation
Journal of Microbiology and Biotechnology. 2014. Jun, 24(6): 756-764
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : September 28, 2012
  • Accepted : March 13, 2014
  • Published : June 30, 2014
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About the Authors
Mame Birame Basse
Jigui Wang
Fuxian Yu
Jiazeng Sun
Zhili Li
Weiquan Liu
weiquanliu8@gmail.com

Abstract
Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in chicken or human tumor cells, localizing in their nuclei as opposed to the cytoplasm of non-transformed cells. The present study was undertaken to investigate whether ABPs1 could potentiate apoptin-induced apoptosis in HeLa cells. ABPs1 and the apoptin genes were successfully cloned into pIRES2-EGFP expression vector and expressed in HeLa cells. We report that ABPs1 augments apoptin cell growth inhibition in a concentration- and time-dependent manner. The DAPI staining and scanning electron microscopy observations revealed apoptotic bodies and plasma membrane pores, which were attributed to apoptin and ABPs1, respectively. Further, ABPs1 in combination with apoptin was found to increase the expression of Bax and to decrease the expression of survivin compared with either agent alone or the control. The apoptotic rate of HeLa cells treated with ABPs1 and apoptin in combination for 48 h was 53.95%. The two-gene combination increased the caspase-3 activity of HeLa cells. Taken together, our study suggests that ABPs1 combined with apoptin significantly inhibits HeLa cell proliferation, and induces cell apoptosis through membrane defects, up-regulation of Bax expression, down-regulation of survivin expression, and activation of the caspase-3 pathway. Thus, the combination of ABPs1 and apoptin could serve as a means to develop novel gene therapeutic agents against human cervical cancer.
Keywords
Introduction
Apoptosis is the physiological process by which unwanted or useless cells are eliminated during development and other normal biological processes. It is essential for ontogenesis, tissue homeostasis, and proper function of the immune system [29 , 43 , 50] . The biochemical machinery responsible for apoptosis is expressed in most, if not all, cells [45] . The process is defined based on changes in cellular morphology and biochemical features, including DNA fragmentation, cytoplasm vacuolation, plasma membrane blebbing, and apoptotic body formation [22] .
The chicken anemia virus (CAV) belongs to the genus Gyrovirus of the family Circoviridae. It induces apoptosis in infected tissues of the avian host, resulting in depletion of thymocytes and erythroblastoid cells in bone marrow [1] . The death of infected cells is caused by apoptin, a 14 kDa virally encoded and proline-rich protein, which has no homologous cellular counterparts [15 , 31 - 33 , 36 , 37 , 42 , 46] . One unique feature of apoptin is its ability to cause selective apoptosis in tumor cells, but not in primary, nontransformed cells [13 , 20] . Currently, apoptin might be one of the most interesting lead molecules for antitumor therapy development and be useful to delineate the mechanisms of oncogenic transformation. In recent years, drug combinations have gained considerable attention because of their beneficial effects in overcoming intrinsic tumor cell resistance to apoptosis. Therefore, finding new combination treatment strategies for synergistic therapeutic effects is desirable.
Antibacterial peptides (ABPs) are small cationic molecules that are part of the nonspecific defenses against bacteria, controlling bacterial infections and coordinating host responses to infection in many animal systems [6] . Cecropin B, which belongs to the cecropin family of ABPs, was originally isolated from the giant silk moth Hyalophora cecropia [21] . It is known that the peptide acts by causing the formation of membrane defects or “pores” [11] . It possesses high antibacterial and antiproliferative activities, and is effective against a wide range of both gram-positive and gramnegative bacteria in micromolar concentrations [9 , 10 , 25 , 47] . Cecropin B mutant, with 4 kDa molecular mass [48] was used in this study. Bax, a pro-apoptotic protein belongs to the Bcl-2 gene family and it promotes apoptosis by competing with Bcl-2 proper. It is believed to induce the opening of the mitochondrial voltage-dependent anion channel [35] . This results in the release of cytochrome c and other pro-apoptotic factors from the mitochondria often referred to as mitochondrial outer membrane permeabilization, leading to activation of caspases. Survivin is a member of the inhibitors of the apoptosis gene family, which is involved in the control of cell division and the inhibition of apoptosis. This protein, which is expressed in the most common human cancers, exerts its anti-apoptotic activity and chemoresistance by interfering with the processing and activity of caspases [4 , 40] . The morphological and nuclear changes associated with apoptosis are typically caused by the sequential activation of a family of cysteinyl-aspartate-specific proteinases called caspases [34 , 41] .
Caspase-3, the main executioner caspase, can be activated by the initiator caspase, caspase 8, following activation of cell-surface death receptors in the extrinsic apoptotic pathway. Alternatively, the intrinsic apoptotic pathway can activate caspase-3 following the release of pro-apoptotic factors from the mitochondria and activation of the initiator caspase, caspase 9. In this study, we investigated our hypothesis, whether ABPs1 combined with apoptin could augment apoptin-induced apoptosis in HeLa cells. The results presented in the current study suggest that ABPs1 in combination with apoptin enhanced apoptin-induced apoptosis in vitro , which was correlated with the downregulation of survivin and up-regulation of Bax and caspase-3 activities.
Materials and Methods
- Cells and Reagents
The HeLa cells were obtained from the American Type Culture Collection (ATCC). HeLa cells were cultured with RPMI 1640 (Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated with 5% CO 2 at 37℃. The medium was supplemented with 10% FBS, 100 units/ml penicillin, and 100 mg/ml streptomycin. The cytomorphological changes were observed by staining cells with DAPI (Invitogen). The broad-range caspase inhibitor DEVD-FMK and the colorimetric caspase substrate Ac-DEVD-pNA were sourced from MBL (Medical & Biological Lab, Japan), whereas MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diplenyltetrazolium bromide) and propidium iodide (PI) were acquired from Sigma (Sigma-Aldrich, St. Louis, MO, USA).
- cDNA, Vectors, and Transient Transfection
ABPs1 was obtained from Dr. Yu [48] and apoptin was procured from the Changchun Military Medical Institute (China). Apoptin and ABPs1 cDNA were inserted into the Eco R1 and Bam H1 cloning sites of pIRES2-EGFP vector (Clontech, Palo Alto, CA, USA). Successful cloning was confirmed by restriction digestion and sequence analysis. Transfections were performed using lipofectamine reagent according to the manufacturer’s instructions (Invitrogen).
- Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis and Quantitative Real-time RT-PCR
Total RNA was isolated with the reagent TRIzol (Invitrogen) following instructions provided by the manufacturer. RNA concentrations were determined by NanoDrop. We reverse-transcribed 1 μg of total RNA using a cDNA kit (TianGen, Beijing, China). Each reverse transcript was amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. RNA templates were used to generate cDNAs for survivin, Bax, and GAPDH by PCR. The PCR primers were designed and used as shown in Table 1 . RT-PCR was performed by a DNA thermal cycler (Bio-Rad, Hercules, CA, USA). The PCR products were visualized in 2% agarose gels with ethidium bromide staining under UV transillumination. Fluorescent PCR analysis was performed using the Bio-Rad iCycler 5 (Bio-Rad). RNA was amplified by qPCR in 20 μl reactions using SYBR1 Premix Ex Taq (TAKARA Biotechnology, Dalian, China) with 1 μl of each primer. Assays were performed in triplicate and analyzed using an ABI 7900HT-sequence detection system (AB Applied Biosystems, San Mateo, CA, USA). The gene expression levels obtained were normalized by mRNA expression of GAPDH. The relative mRNA was then presented in relation to the control.
Primers used in this study for amplification of apoptin and ABPs1 and for RT-PCR and qPCR of survivin, Bax, and GAPDH.
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a(VP3) Viral Protein 3; b(ABPs1) Antibacterial Peptide s1; c(Surv.) Survivin; d(GAPDH) Glyceraldehyde-3-Phosphate Dehydrogenase; F: Forward; R: Reverse.
- Cells Death Assessment and DEVDase Assay
Cells were transfected with apoptin and ABPs1 and the two-gene combination. After the indicated time, post-transfection cells were fixed with 3% formaldehyde in PBS, stained with DAPI, and examined microscopically. For the flow cytometry analysis, cells were harvested, fixed in 70% ethanol, and stained with PI (40 μg/ml final concentration) in phosphate buffered saline solution containing RNAse (TransGen Biotech, China) and samples were processed and screened using FACSCalibur. The data were analyzed with CellQuest Pro software (BD Biosciences, Korea). The cytotoxicities of the cell-permeable apoptin, ABPs1, the twogene combination, and the control were measured using the MTT assay [18] . The membrane defects and the cytoplasmic projection of HeLa cells were observed by scanning electron microscopy. Fixation of the cells in the coverslip was carried out in 2.5% glutaraldehyde followed by postfixation in 1% osmium tetroxide. The samples were dehydrated in alcohol over silica gel. The final embedding was in isoamyl acetate. The coverslip was shattered from the polymer plug by immersing the vial in liquid nitrogen. Specimens were ion sputtered by Eiko IB-3, and examined using a scanning electron microscope (Hitachi S-3400N, Japan). Colorimetric protease assay of caspase-3 activity (DEVDase assay) was assayed according to the manufacturer’s instructions (Medical & Biological Lab) using the colorimetric caspase substrate Ac-DEVD- p NA (2 mM). The colorimetric release of p -nitroaniline from the Ac-DEVD- p NA substrate was measured using a light wave of 405 nm with an ELISA reader (BIO Tek ELx800, Winooski, VT, USA).
- Statistical Analysis
Results are presented as the mean ± SD. ANOVA (Tukey’s test) was used to analyze the data obtained after treatment with ABPs1, apoptin, and the two-gene combination. Values were regarded significant at * p < 0.05; all error bars represent standard deviation (SD).
Results
- Cytomorphological Effects of ABPs1 and Apoptin Expression on HeLa Cells
The transfection efficiency was evaluated by calculating the percentage of GFP-positive cells, which was high ( Figs. 1 A and 1 D). The expression effect was studied by analyzing the cytomorphological changes using DAPI staining. Cells were transfected with pIRES2-EGFP-ABPs1, pIRES2-EGFP-apoptin, and the two-gene combination plasmids ( Figs. 1 F and 1 H), or with control plasmid ( Fig. 1 E). DAPI staining revealed apoptotic changes of formation of rounded apoptotic bodies in cells transfected with pIRES2-EGFP-apoptin and the two-gene combination plasmids. The study clearly confirmed, by DAPI staining, that ABPs1 and apoptin were expressed and that they induced morphological changes. No such changes were seen in control cells.
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Apoptotic changes observed with expression of ABPs1, apoptin, and the two-gene combination in HeLa cells. (GFP) Fluorescence indicated expression of the genes and apoptotic changes at 48 h post-transfection with ABPs1 (B), apoptin (C), and two-gene combination plasmids (D) or control vector pIRES2-EGFP (A). (DAPI) Cells were fixed and stained with DAPI 48 h post-transfection. Arrows indicate apoptotic changes, nuclear condensation, and formation of rounded apoptotic bodies, in cells transfected with pIRES2-EGFP-ABPs1 (F), pIRES2-EGFP-apoptin (G), and the two-gene combination plasmids (H), along with control vector pIRES2-EGFP (E).
- Effect of ABPs1 and Apoptin on Cell Viability
To examine the effects of ABPs1 and apoptin on HeLa cell growth, cells were transfected with increasing concentrations of ABPs1, apoptin, or the two-gene combination, for 24, 48, and 72 h. As shown in Fig. 2 A, ABPs1 transfection inhibited cell growth in a time-concentration-dependent manner. At Δ72 h, cell growth showed a 3-fold asymmetric inhibition by ABPs1 compared with Δ48 h ( Fig. 2 A). These results indicated that ABPs1 alone was an effective inhibitor of HeLa cell growth. The effect of apoptin on cell growth was evaluated, and we found a symmetric (Δt) inhibition of cell growth. In a concentration-dependent manner, cells expressing ABPs1 in different concentrations (0.2, 0.4, and 1 μg/ml) exhibited decreases, or similar percentages of cell viability. However, cells expressing apoptin and the two-gene combination with increasing concentrations revealed only decreases of cell viability, which was more effective in cells treated with 1 μg/ml of the two-gene combination ( Fig. 2 C). Further studies were undertaken to investigate whether or not cells expressing the two-gene combination were more sensitive to the cytotoxic effect of apoptin.
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Effect of ABPs1, apoptin, and the two-gene combination on HeLa cells viability by MTT assay. The time- and concentration-dependent cell viability was measured by treatment of serially diluted plasmid DNAs (0.2, 0.4, and 1 μg/ml) for ABPs1 (A), apoptin (B), and the mixture ABPs1 and apoptin (C) for 24, 48, and 72 h. MTT reagent was added into growing cells (96 well culture plates) and incubated for 2 to 4 h until the purple precipitation was visible. Detergent reagent was added and the plate was left at room temperature in the dark for 2 h. The resulting purple formazan was quantified by spectrophotometric means (570 nm). Data are presented as the mean ± SD; *P < 0.05 based on repeated measure ANOVA (Tukey’s test) (n = 3).
- ABPs1 Augments Apoptotic Effect of Apoptin
To investigate possible synergistic effects of ABPs1 and apoptin, propidium iodide staining, detected by flow cytometry, was used to detect cells undergoing apoptosis ( Fig. 3 ). In accordance with this method, HeLa cells were evaluated by flow cytometry 48 h post-treatment with ABPs1 and apoptin alone, or in combination. All data were analyzed by CellQuest Pro software (BD Biosciences).
The rate of cells undergoing apoptosis was obtained 48 h post-transfection. As shown in Fig. 3 , the apoptotic rates of cells with ABPs1 transfection were 47.52%, 50.45% with apoptin, and 53.95% with the two-gene combination. We found that the number of cells undergoing apoptosis was greater in the cells with a combination of apoptin and ABPs1 transfection compared with the control and apoptin alone.
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Potentiation effect of apoptin by ABPs1 on the apoptotic rate of HeLa cells. After 48 h, cells were harvested, fixed in 70% ethanol, and stained with PI (40 μg/ml final concentration) in PBS solution containing RNAse and samples were processed and screened using FACSCalibur. M1 represented cells undergoing apoptosis. Data were analyzed with CellQuest Pro (n = 3).
- Effect of ABPs1 Expression on the Regulation of Bax and Survivin in HeLa Cells
To investigate whether ABPs1 could induce cell death by utilizing apoptosis pathways, we evaluated the relative mRNA of the pro-apoptotic gene Bax and the anti-apoptotic gene survivin in HeLa cells after transfection. As shown in Fig. 4 A, transfection of HeLa cells with ABPs1 induced a quantitative increase in the level of Bax expression. ABPs1 induced quantitative inhibition of survivin expression ( Fig. 4 B). Furthermore, PCR analysis showed similar changes in the Bax and survivin expression levels produced by ABPs1, suggesting that ABPs1 modulates Bax and survivin expression at the transcriptional level.
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Abrogation of constitutive and induced Bax and survivin mRNA by ABPs1. Total RNA were extracted from cells after transfection with ABPs1, apoptin, and two-gene combination for 24 h, and Bax and survivin expression levels were evaluated as described in Materials and Methods. (A) ABPs1 augments Bax expression in HeLa cells. (B) Survivin expression is down-regulated by ABPs1 in HeLa cells. Data are presented as the mean ± SD of three independent experiments (n = 3); *P < 0.05, by comparison with the respective group.
- Effects of ABPs1 on the Plasma Membrane and Caspase-3 Activity in HeLa Cells
To investigate whether ABPs1 could cause membrane defects and induce apoptosis by caspase-3 activation, HeLa cervical cells were transiently transfected with ABPs1. At 48 h, post-transfection cells were examined microscopically for morphological changes and the effects of ABPs1 on caspase-3 activity. Cells were sensitive to ABPs1 and showed membrane defects ( Fig. 5 B). In contrast, cells transfected with apoptin and the control showed no signs of membrane pores. To investigate the effect of ABPs1 on caspase-3 activity, cells were transfected with ABPs1 and compared with untreated cells. The DEVDase activity of apoptotic cells among ABPs1-transfected cells was determined at 48 h. Cell extracts were prepared and incubated with the colorimetric caspase-3 substrate Ac-DEVD- p NA. A 3.5-fold increase of caspase-3 activity could be observed in ABPs1-transfected cells as compared with untreated cells and apoptin, respectively ( Fig. 6 ).
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Morphological changes induced by ABPs1 potentiate apoptin-induced apoptosis in HeLa cervical cells. Cells transfected with ABPs1, apoptin, and the two-gene combination were harvested, fixed, and subsequently washed in PBS as described in Materials and Methods. Cells were then stained with isoamyl acetate. The pictures were taken 48 h post-transfection. Arrows show membrane “pores”.
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Potentiation of apoptin-induced caspase-3 activation by ABPs1. Cells were treated with ABPs1, apoptin, and the two-gene combination. At 48 h post-transfection, cell lysates were assayed for caspase-3 activity as described in Material and Methods. Data are presented as the mean ± SD of three independent experiments (n = 3); *P < 0.05, by comparison with the respective group.
Discussion
In recent years, novel approaches for sensitizing cancer cells to be killed with microbial gene compounds have drawn considerable attention. The present study was undertaken to investigate whether the ABPs1 cecropin Blike protein could potentiate apoptin-induced apoptosis in HeLa cervical cancer cell lines. The first concern regarding apoptin as an antitumor drug is that it is localized at the nucleus of tumor cells and induces G2/M arrest and apoptosis. The second concern regarding ABPs1 as an antitumor drug is that the peptide acts by causing membrane “pores” in gram-positive and gram-negative bacteria in micromolar concentrations [7 , 26] , but it is also active against certain fungi [14] , metazoan [9] , and protozoan parasites [16] .
The third concern regarding ABPs1 and apoptin in combination as an antitumor drug is that they may induce higher levels of apoptosis in cancer cell lines. In this study, pIRES2-EGFP-ABPs1 and pIRES2-EGFP-apoptin plasmids were successfully constructed. The morphological characterization showed that ABPs1 causes membrane pores in HeLa cells ( Fig. 5 B). Several groups have reported that the ABPs1 amino acid composition, amphipathicity, cationic charge, and size allow them to attach to and insert into membrane bilayers to form pores by “barrel-stave,” “carpet” or “toroidalpore” mechanisms [8 , 11 , 39] .
However, treatment with apoptin did not induce membrane “pores” on HeLa cells, but decreased the plasma membrane microvilli ( Fig. 5 C) compared with the control. Furthermore, the combination of ABPs1 and apoptin showed in addition to membrane “pores,” a decrease of cell microvilli, as well as deformation and loss of contact to neighboring cells ( Fig. 5 D).
The changes in the number of the plasma membrane microvilli observed in cells treated with ABPs1 and apoptin separately were similar. It has been reported that the presence of a large number of microvilli on the top surface of cultured mammalian cells may play a role in functions such as hemadsorption [30] , cell aggregation [27] , and cell fusion [19] .
DAPI staining revealed cytomorphological changes of nuclear condensation in cells transfected with apoptin and the two-gene combination ( Figs. 1 C and 1 D). Similar characteristic morphological changes of apoptosis have been reported [23 , 24 , 44] . The results revealed in terms of cytomorphological and plasma membrane changes that ABPs1 potentiates apoptin-induced apoptosis in cells. The cell viability assay and the PI staining study indicated that ABPs1 combined with apoptin enhanced noticeable cell growth inhibition ( Fig. 2 C) and increased cells undergoing apoptosis ( Fig. 3 ), compared with either agent alone or the control. In a time-concentration-dependent manner, ABPs1 and apoptin induced, asymmetrically and symmetrically, loss of the cells, respectively ( Fig. 2 A). The combination of the two genes was more effective than cells treated separately with ABPs1 and apoptin. In a concentration-dependent manner, 0.2 μg/ml of ABPs1 was effective in inhibiting cell growth, and even when the concentration was increased the effect on the cell viability was quite similar ( Fig. 2 A). The increase of apoptin concentration was correlated with the loss of viable cells and this effect was higher when the two genes were combined. These results indicated that ABPs1 in small doses was effective in inhibiting cell growth by causing membrane defects, and the increase of apoptin concentration was necessary to obtain less viable cells. In addition, the flow cytometry analysis showed that ABPs1 potentiates apoptin-induced apoptosis ( Fig. 3 ).
The transcriptional changes of Bax and the survivin genes as an apoptosis inducer and inhibitor, respectively, and the activation of caspases-3 as an executioner of apoptosis, in response to ABPs1, apoptin, and the two-gene combination expression, were investigated.
Our results revealed that the expression of Bax increased in cells expressing ABPs1 and apoptin in combination ( Fig. 4 A). One of the decisive factors for programmed cell death is the Bcl-2 protein family. In particular, Bax and Bcl-2 are the major apoptotic proteins in this family and act as apoptotic inducers and inhibitors, respectively [12 , 38] . As a result, cytochrome c stimulates caspases-3 that directly promotes apoptosis [2] .
Moreover, our results showed that cells expressing ABPs1 and apoptin in combination decreased the transcription of survivin ( Fig. 4 B), which potentiates the expression level of caspase-3 protein ( Fig. 6 ), compared individually and with the control. In addition, cells with ABPs1 transfection showed an increase of caspase-3 activity compared with cells expressing apoptin.
The prosurvival molecule survivin, a member of the inhibitor of the apoptosis protein family, has been implicated in the control of cell division and apoptosis [3 , 5] . Survivin’s anti-apoptotic function is executed via its ability to prevent caspase activation. Growing evidence suggests that survivin is responsible for drug resistance in cancer cells [28 , 49] . In summary, our current findings showed that ABPs1 potentiates apoptin-induced apoptosis by increasing caspase-3 activity levels in HeLa cervical cancer cell lines. Membrane defects, up-regulation of Bax mRNA, and down-regulation of survivin mRNA might play a role in caspase-3 activity. However, further studies are necessary to understand the optimal dose and delivery method before clinical applications can be considered.
Acknowledgements
We would like to thank Dr. Zou Qiang for the helpful assistance given, and Dr. Mamadou Aliou Diallo for critically reading the manuscript. Jim Hesson of AcademicEnglishSolutions.com revised the English. Our work was supported by grants from the National Transgenic Engineering Foundation of China (No. 2009ZX08006-010B) and the Chinese Scholarship Council in collaboration with the China Agricultural University.
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