Chemicals
Ellipticine was purchased from Sigma (St. Louis, MO, USA). The available synthetic ellipticine is of more than 98% purity. A stock solution of 10 mM in DMSO was stored at −20°C, and freshly dissolved in media. The DMEM medium, penicillin and streptomycin antibiotic mixture, sodium pyruvate and glutamine supplements were obtained from Sigma (St. Louis, MO). Fetal bovine serum was acquired from Invitrogen (Grands Islands, NY).
Transfection of constructs
Cells grown on sterile histologic slides for 24 h were transfected with a 2 μL mixture of LipofectAMINE (Invitrogen) and Akt or green fluorescent protein (GFP)-tagged light-chain 3 (LC3) plasmid (GFP-LC3) in Opti-MEM medium (Invitrogen) at a ratio of 1/1 in volume. After 6 h of incubation, cells were placed in regular complete medium and cultured for 24 h. As cells were treated with ellipticine or vehicle control for the time specified, the medium was changed to regular media, and cells were further incubated in 2 % serum-supplemented media for 24 h at 37°C.
The slides were washed with PBS, and cells fixed in cold methanol. Cells were washed in PBS twice, and coverslips were mounted with glycerol/PBS (3:1) solution.
Slides were examined under a fluorescent microscope (Leica, Singapore).
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Immunofluorescence analysis
The cells grown on coverslips were fixed for 10 min in PBS containing 3.7 % para-formaldehyde. The fixed coverslips were washed in PBS containing 0.1 % Triton X-100 for 10 min, washed twice in PBS (5 min), and incubated in a blocking buffer (PBS containing 0.2 % BSA) for 30 min. The cells were then incubated in the blocking buffer containing the primary antibody for 1 h and washed three times in PBS (5 min) before incubating with the appropriate TRITC-conjugated secondary antibody plus DAPI (Molecular Probes, Grand Island, NY, USA) for further 30 min.
The cells were washed three times in PBS (5 min) and washed in water. The stained cells were mounted on glass slides and examined for fluorescence under a fluorescent microscope (Leica).
Results
The suppressed cell viabilities and increased sub-G1 cell populations can be reverted by dominant-negative AktS473A
The apoptotic cell death in tumor cells is regulated by tumor suppressor p53 [69].
Collaboration of nuclear p53 and Akt resulted in growth inhibition by ellipticine in A549 cells [70]. It is known that AktS473 or AktT308 are critical sites in orchestrating Akt activity. To know more on how phosphorylated Akt affects cell growth, A549 cells were transfected with construct of AktS473A and AktT308A, respectively. They were obtained by converting the phosphorylationsite, S473 or T308, of the regulatory determinants in the encoded Akt sequence to alanine by site mutation. The cells were treated with ellipticine, harvested, and counted following plasmid transfection. The
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results showed that A549 cells transfected with construct AktS473A became less
sensitive to the drug after 48 h treatment, as compared with those of AktT308A (Fig. 1a).
Thus, A549 cells transfected with construct of dominant-negative AktS473A, but not that of AktT308A, were rescued from growth inhibition by ellipticine. To learn how cell cycle population distributions were affected by ellipticine, the collected cells were analyzed by PI-stained flow cytometry. The sub-G1 phase population as induced by ellipticine in A549 cells was suppressed when transfected with AktS473A, but not with AktT308A (Fig. 1b).
AktS473A abrogated ellipticine sensitivity in H1299 cells with ectopic p53
To find out the significance of p53 in ellipticine-induced cell death, p53-null H1299 cells were transfected with construct encoding full-length p53 of either
wild-type (H1299/p53) or the mutant form (H1299/p53R267L) and the respective stable cell lines established. Like A549 cells, the selected clones expressing wild-type p53 exhibited dosedependent growth inhibition by ellipticine. The stable H1299/p53 cells were sensitive to drug treatment as compared to H1299/p53R267L and H1299 cells. As H1299/p53 cell line was transfected with AktS473A, sensitivities toward ellipticine were markedly suppressed. Thus, introduction of AktS473A in both A549 and H1299/p53 cells can revert the induction of cell death. On the other hand, H1299/p53 clone transfected with AktT308A continued exhibiting dose-dependent sensitivity toward the drug (Fig. 2a), that is similar to A549 cells. Both insensitive H1299/p53R267L and H1299 cells were unaffected when transfected with AktS473A. Thus, p53-null cells with ectopic p53 expression became sensitive to ellipticine and dominant-negative AktS473A abrogated cell death in NSCLC cells harboring wild-type p53. Similar to A549 cells, the increased sub-G1 populations in the stable H1299/p53 cells by ellipticine were
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dose-dependent that differed from those of both H1299/p53R267L and H1299 cells, in which only G2/M cells were collected as drug concentrations were elevated (Fig. 2b).
The results altogether proved that the reduced cell viabilities by ellipticine was accomplished by collaboration of p53 and phosphorylated Akt473.
Dominant-negative AktS473A inhibited ellipticine induced nucleus translocation of p53 and Akt in A549 cells
Both Akt and p53 were found moved into nucleus when treated with ellipticine.
The results through staining cells with p53, Akt1/2, and phospho-specific AktS473 antibodies showed localization of merged nucleus p53 and Akt as phosphorylated at S473 in A549 cells. Translocation of nucleus Akt was suppressed when transfected with AktS473A construct as shown by immunostaining with Akt1/2 and p53 antibodies, respectively (Fig. 3a). On the other hand, the nucleus p53 as induced was also
affected when transfected with dominant-negative AktS473A. The nuclear Akt was unaltered after ellipticine treatment following transfection with Akt or AktT308A but vanished with AktS473A as determined by their relative levels statistically (Fig. 3b). In addition, the induced phosphorylated nucleus Akt disappeared as proved by staining with phospho-specific AktS473 antibody upon transfection of AktS473A (Fig. 3c). To strengthen the notion that Akt is moved into the nucleus by drug treatment, extracts of both cytoplasm and nuclear fractions were analyzed by Western blot. The proteolysis cleavage of the precursor PARP serves as marker of genomic integrity signaling the ultimate tenacity to apoptosis [36]. In drug-treated A549 cells, the appearance of an intense nuclear 89-kDa PARP fragment served as a benchmark of apoptotic death (Fig.
3d). The induced fragmentation of PARP was suppressed in cells when transfected with AktS473A construct followed by ellipticine treatment. The results differed from
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A549 cells introduced with Akt and AktT308A, in which Akt was detected remaining in nucleus. Likewise, the nuclear p53 and the corresponding downstream regulator p21 as induced dissipated in cell lysates when transfected with AktS473A construct as shown in Western blot analysis. The data strongly suggested that the presence of
phosphorylated Akt473 in nucleus is critical in driving A549 cells to growth
suppression affected by ellipticine. The progression of the induced cell death involved coordination of nuclear phosphorylated Akt473 and p53 and the effect was knocked down by dominant-negative AktS473A.
Knock-down of p53 inhibited ellipticine-induced nucleus translocation of Akt
Sensitivities toward ellipticine were suppressed in A549 cells transfected with small-hairpin RNA of tumor suppressor p53 (p53 shRNA) as compared with those of control non-specific shRNA (NS shRNA) (Fig. 5a). When nucleus movement of Akt was impaired, the increased PARP cleavage in A549 cells no longer appeared and the intrinsic phospho-Akt was dissipated when transfected with shRNA p53 plasmid prior to drug treatment (Fig. 5b).
The enhanced autophagy during ellipticine-induced apoptosis was deactivated by AktS473A
Genotoxic stress-induced apoptotic cell death can be motivated by autophagy [71]. The variation of microtubule associated light-chain LC3 as a vital element via autophagosome is a good indicator to track the progression of autophagy. Usage of the GFP-tagged LC3 plasmid is an effective method in autophagy determination.
A549 cells were co-transfected with construct encoding GFP-LC3 and various Akt
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plasmids and the distribution of GFP puncta determined following drug treatment.
The emerged punctuate green fluorescence after 24 h treatment of ellipticine in A549 cells suggested correlation between autophagy and the ensued apoptosis (Fig. 6a). The appearance of green punctate fluorescence was diffused in cells when transfected with GFP-LC3 construct together with AktS473A. What is more, the puncta remained intact in cells once Akt or AktT308A was transfected altogether, suggesting that the apoptotic cell death was attributed to autophagy. Moreover, the increased LC3-II/LC3-I ratio was reduced by AktS473A, but not by Akt and AktT308A. The results indicated the importance of p53 and Akt coorporation in elaborating autophagy-associated cell death and growth inhibition by ellipticine (Fig. 6b).
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Figures and legends
Figure 1. (A) Cell viabilities of A549 cells as treated with ellipticine following various Akt transfection. Cells were seeded onto 6-well plates (5 ×103 cells/well).
After 24 h for complete adherence, the cells were transfected with various Akt constructs and incubated with vehicle control (0.2 % DMSO) or ellipticine of different concentrations (2 and 5 μM). After 48 h of treatment, the cells were trypsinized and counted by trypsin-exclusion assay. (B) Cell cycle histograms analysis of ellipticine-treated A549 cells. Exponentially growing 3 × 105 A549 cells as transfected with various Akt constructs were cultured with ellipticine (5 μM) (+) or
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without (-) for the time points as indicated. The trypsinized cells were analyzed by flow cytometry. The percentage distribution of cell cycle phases was determined by FACS analysis following PI staining. The error bars represented standard errors in three independent experiments conducted.
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Figure 2. Cell cycle histograms analysis of ellipticine-treated cells. (A) Exponentially growing 5 × 103 H1299, H1299/p53 and H1299/p53R267L cells
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transfected with various Akt constructs were cultured without (–) and with ellipticine (2 and 5 μM) (+) for 48 h. The trypsinized cells as collected were counted by
trypsin-exclusion assay. (B) Cell cycle histogram. The exponentially growing cells of H1299, H1299/p53, and H1299/p53R267L (5 × 103) were cultured with ellipticine (2 and 5 μM) or 0.2 % DMSO control for 48 h. The collected trypsinized cells were analyzed by flow cytometry. The percentage distribution of cell cycle phases was determined by FACS analysis following PI staining. The error bars represented standard errors in three independent experiments as conducted.
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Figure 3-1. (A) Immunofluorescence analysis. Cells plated on coverslips in 6-well plates were treated with the presence (+) or absence (-) of ellipticine after being transfected with various Akt constructs for 48 h. The cells were fixed and stained with Akt (green) and p53 antibody (red), respectively. The cells were counterstained with DAPI to visualize the nuclei (blue). (scale, 10 μm) (B) Statistical analysis.
The presence of Akt in the nucleus was estimated following immunostaining after ellipticine treatment in cells transfected with different Akt constructs. By counting 100 cells, the stained cells with nuclear Akt were observed under each condition, and the converted percentages were obtained and compared.
The error bars represented standard errors in three independent experiments as conducted. (**p\0.01).
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Figure 3-2. (C) Immunofluorescence analysis. The A549 cells after being
transfected with various Akt constructs were cultured in the presence (+) or absence (-) of ellipticine as specified in (A). The cells were fixed and stained with pAkt473 (green) and p53 antibody (red), respectively. The cells were then counterstained with DAPI to visualize the nuclei (blue). (scale, 10 μm) (D) Western blot analysis. An equal
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amount of proteins from both nuclear and cytoplasmic fractions of A549 cells as being transfected with various Akt constructs were cultured in the presence (+) or absence (-) of 5 μM of ellipticine and separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in fresh blocking solution and probed for 1 h with 1:3000 dilution of PARP, MDM2, Akt1/2, phosphorylated-Akt473, p21, p53, a-tubulin or Histone H1 antibody, followed by incubating with a 1:4000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system. The numbers underneath signified relative intensities compared with the results of vehicle control 0.2 % DMSO and transfection with the empty vector.
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Figure 4-1. (A) Immunofluorescence analysis. H1299/p53 cells were treated with 5 μM of ellipticine after being transfected with various Akt constructs for 48 h. The cells were fixed and stained with Akt (green) and p53 antibody (red), respectively.
The cells were counterstained with DAPI to visualize the nuclei (blue). (scale, 10 μm) (B) Statistical analysis. The presence of Akt in the nucleus was estimated following
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immunostaining after ellipticine treatment in cells transfected with different Akt constructs. By counting 100 cells, the stained nuclear Akt was observed under each condition, and the converted percentages were obtained and compared. The error bars represented standard errors in three independent experiments conducted. (**p＜0.01).
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Figure 4-2. (C) Western blot analysis. An equal amount of proteins from both nuclear and cytoplasmic fractions of H1299/p53 cells as being transfected with various Akt constructs were cultured in the presence (+) or absence (-) of ellipticine and separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in fresh blocking solution and probed for 1 h with 1:3000 dilution of PARP, MDM2, Akt1/2, phosphorylated-Akt473, p53, α-tubulin, or Histone H1 antibody, followed by incubating with a 1:4000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system. The numbers underneath signified relative intensities compared with the results of vehicle control 0.2 % DMSO and transfection of the empty vector.
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Figure 5. (A) Cell growth determination of A549 cells as affected by p53 shRNA.
The A549 cells were transfected with either p53 shRNA or NS shRNA and cultured with ellipticine for 48 h. Cells were trypsinized and the viable cells counted by trypan blue exclusion assay. (B) Western blot analysis. A549 cells cotransfected with p53 shRNA and different Akt constructs were treated with 5 μM of ellipticine for 48 h. An
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equal amount of proteins from both nuclear and cytoplasmic fractions was separated by SDS-PAGE separating gel and electro-blotted. The blots were then incubated in blocking solution and PARP, MDM2, Akt1/2, phosphorylated-Akt473, p53, α-tubulin and Histone H1 antibody followed by a 1:4000 dilution of horseradish
peroxidase-conjugated secondary antibody and then developed by ECL detection system. The numbers underneath signified relative intensities compared with the results of transfection without p53 shRNA transfection and 0.2 % DMSO treatment.
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Figure 6. (A) Autophagosome regulation as visualized in GFP-LC3 expressing A549 cells by ellipticine. A549 cells co-transfected with various Akt constructs and GFP-LC3 plasmid were incubated with vehicle DMSO control or 5 μM of ellipticine and observed under the fluorescence microscope for GFP (green) and counterstained with DAPI (blue). The green punctuate fluorescence (arrow) in GFP-LC3 transfected cells signaling autophagy formation was assessed. (B) Western blot analysis. An equal amount of protein lysates from A549 cells as co-transfected with various Akt constructs and GFP-LC3 plasmid followed by treatment with ellipticine for 24 h were separated by SDS-PAGE separating gel and electro-blotted. The blots were incubated
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in blocking solution and LC3 and GAPDH antibody, respectively, followed by a 1:4000 dilution of horseradish peroxidase-conjugated secondary antibody and then developed by ECL detection system.
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Discussion
Tumor suppressor p53 is an important effector in promoting apoptosis under genotoxic stresses. As a promising antitumor agent, ellipticine activated p53 and caused apoptotic death in human cancer cells [72-75]. Ellipticine and its derivatives were known in mediating p53-dependent apoptosis in human breast adenocarcinoma cells, lymphoma, and glioblastoma cell lines [76-78]. The cell growth inhibition was associated with collaboration of p53 and Fas/Fas ligand death receptor [76,79].
Another derivative 7-hydroxyisoellipticine was demonstrated inhibiting proliferation of leukemia cells by preventing cells from entering G2 phase and final mitosis [80].
Previous report showed that ellipticine inhibited ubiquitination and nuclear export of p53 [81]. As an effective topoisomerase II inhibitor of growth in A549 cells,
ellipticine first arrested cells at G2/M check points. The PI3K inhibitor blocked the final apoptotic death by suppressing sub-G1 cell population and increasing viable cells.
Furthermore, ellipticine induced cell death via autophagy and apoptosis in human NSCLC cells by coordinating Akt and p53 [70]. The increasingly arrested A549 cells at G2/M phase as affected by phosphorylated Akt lead to final commitment of
apoptotic cell death. Treatment with PI3K inhibitor alone is sufficient to inhibit Akt activity and cell proliferation and promote apoptosis [82,83]. Despite the potential values of Akt inhibitors as useful cancer therapy, only few of them were known and proved useful. Nuclear Akt acting as an apoptosis stimulator rather than as a repressor is likely the effect of a different set of substrates in the nucleus [84]. However, some reports indicated that treatment of murine hematopoietic cells with topoisomerase inhibitors upregulated phosphorylated Akt, followed by rapid dephosphorylation to its basal level and the drug resistance was increased without influencing cell growth [85].
It is therefore reasonable to find out the roles of the principle regulatory sites of Akt,
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including S473 and T308, in facilitating the apoptotic cell death in A549 cells as a result of ellipticine treatment. The induced drug sensitivity and cell death in A549 cells (Fig. 1a) were abolished as a result of attenuated Akt phosphorylation by transfecting AktS473A, but not by AktT308A (Fig. 3a). As dominant-negative AktS473A affects ellipticine sensitivities, nucleus translocation of Akt and p53 was blocked simultaneously. The results supported previous findings that ellipticine serves as an Akt-dependent cell growth inhibitor, thereby making it an effective drug in lung cancer treatment. Cellular DNA damage as a result of topoisomerase inhibitor treatment causes either irreversible senescence or apoptosis in tumor cells [86]. The emergence of intense 89-kDa fragment of nuclear polymerase PARP is associated with DNA repair and apoptotic cell death. The characteristic PARP cleavage appeared in nucleus serves as an apoptosis marker in response to environmental stress
[37,87,88]. The reaction to topoisomerase inhibitor in carcinoma cells with wild-type p53 can be characterized by p53 and MDM2 activation with apparent apoptosis [69].
Ellipticine inhibited the growth of A549 cells by activating p53 accompanied with cleavage of the nuclear 116-kDa PARP precursor into an 89-kDa fragment after 48 h treatment. The activated Akt can be located to cell membrane in response to
stimulation, in which Akt3 was found localized in the nucleus in the presence of ionizing radiation or epidermal growth factor [89]. It is therefore of great value to find out that topoisomerase inhibitor ellipticine assists in Akt1/2 and p53 nuclear
migration that lead to cell death. After mitogenic stimulation, Akt phosphorylates multiple substrates related to cell cycle progression and lead to reduction of p53 transactivation [90] and the PH domain of Akt is needed for nucleus translocation [91]. The work showed that activation of p53, its downstream effector p21, and MDM2 coincided with concomitant Akt phosphorylation at S473 as activated by ellipticine. The phosphorylated Akt was excluded from nucleus upon AktS473A
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transfection. While ellipticine promoted nucleus translocation and phosphorylation, Akt activation may stabilize p53 without binding to MDM2 and inactivate
ubiquitination and degradation of p53 [90]. Activation of p53 blocks the
anti-apoptotic effects of Akt and leads to apoptosis [92]. Our work corroborates previous findings that nucleus entry of Akt stabilized p53, diminished the roles of MDM2, and induced apoptotic cell death [93]. Activated Akt serves as a
pro-apoptotic marker in Pemetrexed-mediated S-phase arrest and cell death [94]. The work demonstrated that ellipticine enhanced nucleus migration and Akt
phosphorylation at S473 in both A549 cells (Fig. 1) and the stable H1299 cells with ectopic p53 and reduced their growth rate, but not in those introduced with AktS473A (Fig. 4). Since functional PTEN was reportedly active in A549 cells, the
ellipticine-induced phosphorylation of Akt on S473 could be attributed to PTEN activation [71,95]. Activation of Akt promotes nuclear translocation of MDM2, which in return mediated p53 degradation through an ubiquitin-dependent pathway on proteasomes [90]. In addition, p53 and MDM2 are known forming an auto-regulatory feedback loop, and p53 positively modulates MDM2 expression and negatively regulates p53 formation [92,96]. Thus, it is feasible that wild-type p53 and phosphorylated Akt act together in mediating cell death by ellipticine that was abolished by mutant AktS473A. The results could suggest that phosphorylated Akt at S473 down-regulates MDM2 activities and stabilizes nucleus p53 [92]. Previous reports showed that radiosensitization as Akt inhibitor exerted anticancer effects through autophagy activation [97]. The Akt-induced autophagy contributed to the effectiveness in malignant glioma cell treatment [98]. On the other hand, the
autophagy and apoptosis pathways leading to cell death in tumor cells in response to anticancer drugs may act separately or by complementation [99,100]. The deficiencies of autophagy could lead to malignant transformation [101]. Strategies in developing
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Akt inhibitions have been demonstrated effective in restraining cell proliferation as well as developing apoptosis in vitro and in vivo in cancer cells [102]. The two sites of T308 and S473 of Akt can be independently catalyzed and appear to be directed at distinct cellular pathways. Site T308 is essential for regulation of TORC1 and thus protein synthesis, whereas S473 is important for controlling the transcription of genes mediating cell survival and apoptosis [103]. The results showed that regulation at
Akt inhibitions have been demonstrated effective in restraining cell proliferation as well as developing apoptosis in vitro and in vivo in cancer cells [102]. The two sites of T308 and S473 of Akt can be independently catalyzed and appear to be directed at distinct cellular pathways. Site T308 is essential for regulation of TORC1 and thus protein synthesis, whereas S473 is important for controlling the transcription of genes mediating cell survival and apoptosis [103]. The results showed that regulation at