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Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIM-mediated apoptosis in cancer cells
Accepted Manuscript Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIM-mediated apoptosis in cancer cells Zhenxing Liu, Zhichen Shi, Jieru Lin, Shuang Zhao, Min Hao, Junting Xu, Yuyin Li, Qing Zhao, Li Tao, Aipo Diao PII: DOI: Reference:
S0006-2952(19)30050-4 https://doi.org/10.1016/j.bcp.2019.02.012 BCP 13418
To appear in:
Biochemical Pharmacology
Received Date: Accepted Date:
9 November 2018 8 February 2019
Please cite this article as: Z. Liu, Z. Shi, J. Lin, S. Zhao, M. Hao, J. Xu, Y. Li, Q. Zhao, L. Tao, A. Diao, Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIM-mediated apoptosis in cancer cells, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.02.012
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Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIMmediated apoptosis in cancer cells
Zhenxing Liu, Zhichen Shi, Jieru Lin, Shuang Zhao, Min Hao, Junting Xu, Yuyin Li, Qing Zhao, Li Tao, Aipo Diao*
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, State Key Laboratory of Food Nutrition and Safety, Tianjin 300457, China
* Corresponding author at: School of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China. Email address: [email protected] (Aipo Diao)
ABSTRACT The transcription factor forkhead box O 3A (FOXO3A) is a tumor suppressor that promotes cell cycle arrest and apoptosis. Piperlongumine (PL), a plant alkaloid, is known to selectively kill tumor cells while sparing normal cells. However, the mechanism of PL-induced cancer cell death is not fully understood. We report here that an association of FOXO3A with the pro-apoptotic protein BIM (also known as BCL2like 11, BCL2L11) has a direct and specific function in PL-induced cancer cell death. Using HeLa cells stably expressing a FOXO3A-GFP fusion protein and several other cancer cell lines, we found that PL treatment induces FOXO3A dephosphorylation and nuclear translocation and promotes its binding to the 1
BIM gene promoter, resulting in the up-regulation of BIM in the cancer cell lines. Accordingly, PL inhibited cell viability and caused intrinsic apoptosis in a FOXO3A-dependent manner. Of note, siRNAmediated FOXO3A knockdown rescued the cells from PL-induced cell death. In vivo, the PL treatment markedly inhibited xenograft tumor growth, and this inhibition was accompanied by the activation of the FOXO3A-BIM axis. Moreover, PL promoted FOXO3A dephosphorylation by inhibiting phosphorylation and activation of Akt, a kinase that phosphorylates FOXO3A. In summary, our findings indicate that PL activates the FOXO3A-BIM apoptotic axis by promoting dephosphorylation and nuclear translocation of FOXO3A via Akt signaling inhibition. These findings uncover a critical mechanism underlying the effects of PL on cancer cells.
Keywords: FOXO3A, BIM, Piperlongumine, Apoptosis, Cancer cells, Akt
1. Introduction The FOXO3A protein, a member of the forkhead box O (FOXO) transcription factors, plays a pivotal role in the control of various cellular processes, including cell proliferation, cell cycle, metabolism, apoptosis, autophagy, stress resistance and longevity [1-4]. As transcription factors, FOXOs regulate the expression of their target genes via nuclear translocation modulated by post-translational modifications, such as phosphorylation, ubiquitination and acetylation [5]. Growth and survival signals activate Akt, which phosphorylates FOXO3A on its conserved serine and threonine residues. FOXO3A phosphorylation by Akt impairs its DNA binding activity and promotes its interaction with the chaperone protein 14-3-3, sequestering it in the cytoplasm and rendering it inactive [6, 7]. Similarly, glycogen synthase kinase-3 beta 2
(GSK-3β) and Ikappa B kinase (IKK) have also been shown to inhibit FOXO3A activity by direct phosphorylation [8, 9]. In contrast, FOXO3A protein is activated and released from 14-3-3 under oxidative stress through Jun N-terminal kinase (JNK) signaling [2, 7]. In addition, FOXO3A can also be inhibited by the deacetylase sirtuin 1 (SIRT1) by facilitating its ubiquitination and proteasomal degradation [10].
It has been implicated that the FOXO family is associated with cancer development [1]. The deregulation of FOXO functions will cause uncontrolled cell proliferation and accumulation of DNA damage, which results in carcinogenesis. Active FOXO members act as tumor suppressors by promoting cell cycle arrest and apoptosis [6]. Survival analyses showed that low expression of FOXO3A is associated with a poor prognosis of patients with ovarian cancer [11]. Several studies suggest that activation of FOXO3A inhibits tumor growth and survival, including prostate and breast cancer cell lines and acute myeloid leukemia. The depletion of PI3K/Akt signaling causes significant activation of FOXO3A transcriptional activity, resulting in either cell cycle arrest by cyclin-dependent kinase inhibitor p27 Kip1 activation [12], or apoptotic cell death by FOXO3A-dependent protein PUMA or BIM expression [13, 14]. In addition, extracellular signal-regulated kinase (ERK) phosphorylates FOXO3A at Ser425, degrading FOXO3A via the ubiquitin-proteasome pathway, which inhibits its function to induce cell death and thus promotes cell proliferation and tumorigenesis [15]. Recent evidence has implicated that ΔNp63α protein is a direct FOXO3A transcriptional target, and FOXO3A-mediated expression of ΔNp63α inhibits oncogenic PI3K-, Ras-, and Her2-induced tumor metastasis [16]. Moreover, FOXO3A plays an important role in casein kinase 1α (CK1α)-mediated regulation of autophagy in RAS-driven cancers. CK1α phosphorylates 3
FOXO3A to promote its nuclear exclusion, decreasing expression of autophagy genes and limiting autophagic recycling of nutrients. In contrast, inhibition of CK1α activity stabilizes FOXO3A to elevate autophagic flux, which sensitizes oncogenic RAS-driven cancer cells to lysosomotropic agents via hyperaccumulation of ineffective autophagic vesicles [17]. Thus, the finding for compounds that evoke activation and nuclear translocation of FOXO3A is a promising therapeutic strategy for cancer treatment.
Using HeLa cells that stably express a FOXO3A-GFP reporter, we performed a small-molecule screen to discover compounds that can activate FOXO3A. One compound, piperlongumine, is a potent inducer of nuclear translocation of FOXO3A. Piperlongumine (PL), a plant alkaloid derived from the long pepper (Piper longum), exhibits numerous key biological activities [18]. It has been reported that PL could induce pancreatic cancer cell death by inducing intracellular reactive oxygen species (ROS) accumulation [19]. It has been suggested that direct inhibition of ROS-clearance proteins, such as glutathione S-transferase pi 1 (GSTP1) or Thioredoxin reductase 1 (TrxR1), may contribute to PL-induced ROS accumulation in cancer cells [20]. However, the cellular targets and downstream signaling pathways involving in PL stimulation have not been fully defined.
Here, we provide first evidence that PL induces FOXO3A dephosphorylation and nuclear translocation in various cancer cell lines, including cervical, breast and stomach cancer cells, subsequently promoting expression of the pro-apoptotic protein BIM and activation of apoptosis. In contrast, PL-induced cell death is effectively rescued by FOXO3A knockdown. These findings provide a molecular mechanism to the proapoptotic action of PL and implicate FOXO3A as a relevant mediator of the antitumor activity induced by 4
PL.
2. Materials and methods 2.1. Cell culture and reagents The human cervical cancer cell line HeLa, breast cancer cell line MCF-7, gastric cancer cell line MGC803, normal liver cell line L02 and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC). The cells were routinely cultured in Dulbecco’s modified Eagle’s Medium (Gibco, Eggenstein, Germany) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO2 at 37°C. PL (purity: 99.33%) and LY294002 (99.84%) were purchased from Selleckchem (Houston, TX, USA). PL was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 15 mM. Complete media was used in all experiments, and the concentration of the vehicle, DMSO, was 0.1% in final incubations. Control cells were treated with vehicle alone. All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Antibodies against FOXO3A (#12829), p-FOXO3A (#9466), BIM (#2933), cleaved-caspase 9 (#9501), cleaved-caspase 3 (#9664) and Lamin A/C (#4777) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phosphorylated Akt (p-Akt) (sc-514032), Akt (sc-56878) and β-actin(sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit (LK2001) or antimouse secondary antibodies (LK2003) were purchased from Sungene Biotech (Tianjin, China). FITC Annexin V apoptosis Detection Kit and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). 5
2.2. Plasmid construction and stable cell line The full length human FOXO3A gene (GenBank Accession Number NM_001455.3) was amplified by PCR using forward (5’-AACTCGAGATGGCAGAGGCACCGGCT-3’) and reverse (5’TTGGATCCTTGCCTGGCACCCAGCTCTG-3’) primers, and the cDNAs of HeLa cells as template. The PCR fragment of FOXO3A digested with XhoI and BamHI (New England Biolabs, Ipswich, MA, USA) was cloned into pLVX-AcGFP-N1 plasmid (Clontech, Palo Alto, CA, USA) to generate the FOXO3AGFP transfection vector. All constructs were verified by DNA sequencing. Lentivirus particles were generated by transfecting HEK-293T cells with pMD2.G and psPAX2 packaging plasmids and the corresponding backbone plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Virus were collected 48 h post transfection, filtered, and used fresh for infection overnight in the presence of 10 μg/mL polybrene. HeLa cell line stably expressing FOXO3AGFP was obtained by infection with pLVX-AcGFP-FOXO3A lentiviruses and selected in 1 μg/mL puromycin for at least two weeks.
2.3. Nuclear and cytoplasmic fractionation Subcellular fractionation of cells was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, cells were homogenized in cytoplasmic extraction reagent I with subsequent addition of cytoplasmic extraction reagent II, and centrifuged. The supernatants from the centrifugation stage were cytoplasmic fractions. The insoluble pellets were suspended in nuclear extraction reagent, and nuclear proteins were extracted by 6
centrifugation. 2.4. Cell viability and colony formation assay Cells were seeded in 96-well plates at a density of 8 × 103 cells per well, and cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. After exposure of cells to PL, MTT solution (20 μL, 0.5 mg/mL in PBS) was added and incubated at 37°C for 4 h. Then, DMSO (200 μL/well) was added to dissolve the formazan dyes and the absorbance was measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). For the colony formation assay, cells were plated (1500 cells/well) in 6-well plates and cultured for 7 days in growth medium. After PL treatment (15 μM, 48 h), the colonies were stained with crystal violet (0.2% w/v in 2% ethanol) and photographed under phase contrast microscope. The efficiency of colony formation was quantified by ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA).
2.5. Quantitative real-time PCR (qPCR) Total RNA was extracted from cells using a Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RNA was reverse-transcribed into cDNAs using Revert Aid First Strand cDNA Synthesis kit (Thermo Scientific). The qPCR was performed on an ABI Prism 7900HT Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using Fermentas Maxima SYBR Green/ROX qPCR reagents (Thermo Fisher Scientific). The reactions were carried out in a 96-well plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. GAPDH expression was used as an endogenous control to normalize target gene expression by the ΔΔCt method. Primers used were as follows: BIM, 5’ATGGCAAAGCAACCTTCTGA-3’ and 5’-CGCATATCTGCAGGTTCAGCC-3’; GAPDH, 5’7
CAAGGTCATCCATGACAACTTTG-3’ and 5’-GTCCACCACCCTGTTGCTGTAG-3’. 2.6. Cell apoptosis analysis Cells were plated on 60-mm dishes for 12 h, and then treated with PL (15 μM) for 24 h. Cells were then harvested, washed twice with ice-cold PBS, and evaluated for apoptosis by double staining with FITC conjugated Annexin V and PI in binding buffer for 30 min using a FACSCalibur flow cytometer (BD Biosciences, CA, USA).
2.7. ChIP assay ChIP assays were performed in HeLa cells with ChIP-IT Kit (Active Motif, Carlsbad, CA, USA) using an antibody specific for FOXO3A or normal rabbit IgG according to the manufacturer’s protocols. ChIP samples were subjected to PCR experiments to amplify fragment of the BIM promoter region using primers as follows: 5’-CGGCTTAGAAACTCAGGGCA-3’ (forward) and 5’CAGGCTCGGACAGGTAAAGG-3’ (reverse). To examine the strength of antibody binding to FOXO3A-sites, ChIP samples were subjected to qPCR using the same primers. The Cp value of each ChIP sample was normalized to its corresponding input.
2.8. RNA interference To silence FOXO3A gene expression, the following sequences were synthesized (RIBOBIO, Guangzhou, China): the FOXO3A mRNA target sequence 5’-GCACAGAGUUGGAUGAAGU-3’, the siRNA sense strand 5’-GCACAGAGUUGGAUGAAGUUU-3’, and the antisense strand 5’ACUUCAUCCAACUCUGUGCUU-3’. Transfection of siRNA duplexes was performed with 8
Lipofectamine 2000 reagent according to manufacturer guidelines (Invitrogen). Experimental assays were performed 24 h post-transfection. A non-target siRNA (Invitrogen) was used as a negative control.
2.9. Western blot Cell lysates were prepared by extracting proteins with RIPA buffer (Sigma) containing protease cocktail inhibitor (Roche, Basel, Switzerland). Equal amounts of protein samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), the membrane was incubated with primary antibodies diluted in blocking solution (1:1,000) at 4˚C overnight. After washing with TBST, the membrane was incubated for an additional 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase at a dilution of 1:5,000. The protein bands were visualized using an enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The density of the immunoreactive bands was analyzed using ImageJ software.
2.10. Antitumor study in nude mice All animal experiments were approved by the Tianjin University of Science and Technology’s Policy on the Care and Use of Laboratory Animals. Five-week-old female BALB/c nude mice (18~20 g) purchased from Vital River Laboratories (Beijing, China) were used for in vivo experiments. MCF-7 cells were collected and injected subcutaneously (2 × 106 cells in 200 μL normal saline) into the right flank of nude mice. Six animals per group were used in each experiment. When tumor mass reached 5-6 mm in diameter (~7 days), mice were treated with PL at the dose of 8 mg/kg body weight (152 µg of PL in 200 μL normal 9
saline) by intraperitoneal (i.p.) injection once every other day of induction. Normal saline containing equal amount of DMSO was used as the vehicle control. Tumor volumes were measured twice weekly using a vernier caliper, and the volume was calculated according to the formula: π/6 × length × width of the tumor. At the end of experiment, the mice were sacrificed and the tumors were removed and weighed for use in the protein expression studies.
2.11. Immunohistochemistry (IHC) staining The xenograft tumors were fixed in formalin. For immunohistochemical staining, air-dried tissue sections were rinsed in PBS with 0.1% Triton X-100, then incubated with 0.3% hydrogen peroxide for 30 min to quench endogenous tissue peroxidases. After this, the sections were blocked with 10% goat serum for 30 min, incubated with the FOXO3A, p-FOXO3A antibodies or rabbit IgG overnight at 4°C, then labeled with goat anti-rabbit IgG conjugated to HRP. Immunostaining was visualized with diaminobenzidine (DAB) and stained with hematoxylin. Slides were dehydrated through alcohols and xylene and mounted in permount. Slides were photographed with a fluorescent microscope (Olympus BX53, Tokyo, Japan).
2.12. Statistical analysis All plots show individual data and means ± standard deviation (SD). Data were analyzed by unpaired Student’s t-test or one-way analysis of variance (ANOVA), using GraphPad Prism version 5.01 (GraphPad Software). In all cases, P < 0.05 was considered statistically significant.
3. Results 10
3.1. Piperlongumine treatment induces FOXO3A nuclear translocation in cancer cells Using HeLa cells stably expressing a FOXO3A-GFP fusion protein, we found that PL treatment potently induced FOXO3A-GFP nuclear translocation in a dose- and time-dependent manner. The spatio-temporal kinetics of FOXO3A nuclear translocation was analyzed by exposing the cells to 5, 10, 15 and 20 μM PL for 3, 6, 12 and 24 h (data not shown). The localization of this chimaeric protein was revealed by fluorescence microscopy. Significant nuclear accumulation of fluorescent signals were observed after treatment with 15 μM PL for 12 or 24 h, compared to the control (Fig. 1A). LY294002, a cell-permeable and broad-spectrum PI3K family inhibitor that acts on the ATP-binding site of the enzyme, has been shown to block PI3K-dependent Akt phosphorylation and kinase activity [21, 22]. The inhibition of PI3K/Akt signaling using LY294002 can cause FOXO3A translocation from the cytosol into the nucleus [23, 24]. In our study, similar results were also observed upon treatment with LY294002. To further confirm the nuclear translocation of endogenous FOXO3A in response to PL treatment, subcellular fractionation and Western blot analysis were performed in HeLa, MCF-7 and MGC-803 cells. Following treatment with 15 μM PL for 6, 12 and 24 h, nuclear FOXO3A abundance was significantly increased whereas cytoplasmic FOXO3A abundance was significantly decreased in these cell lines (Fig. 1B). Moreover, we examined the effect of PL on the subcellular localization of FOXO3A in normal liver cell line L02. The results showed that PL treatment did not induce FOXO3A nuclear translocation in L02 cells (Fig. 1C and D). These data indicate that PL induces FOXO3A nuclear translocation in cancer cells.
3.2. Piperlongumine treatment elicits upregulation of pro-apoptotic protein BIM To evaluate the role of PL in the transcriptional activity of FOXO3A, then the expression of FOXO3A 11
target genes were analyzed in HeLa, MCF-7 and MGC-803 cells. Western blot analysis demonstrated a strong increase in isoform BimEL protein levels, and detectable bands corresponding to BimL and BimS isoforms following 12, 24 h cell incubation with 15 μM PL (Fig. 2A). Moreover, research has shown that BIM proteins can be degraded via the ubiquitin-proteasome pathway [25]. MG-132, a proteasome inhibitor, dramatically increased the BimEL protein abundance in the presence of PL, compared to MG132 alone (Fig. 2B), suggesting that the BIM protein accumulation is not due to the inhibition of the ubiquitin-proteasome pathway. FOXO3A is known to bind and activate the BIM gene promoter [26]. Real-time PCR analysis revealed that BIM mRNA levels were significantly increased in HeLa and MCF-7 cells treated with 15 μM PL (Fig. 2C). In addition, data from ChIP experiments showed that the strength of FOXO3A binding to the BIM promoter was significantly increased in response to PL treatment, compared to the control (Fig. 2D). These results suggest that PL induces the expression of FOXO3A-BIM axis genes in cancer cells.
3.3. Piperlongumine inhibits cell viability and causes apoptosis in cancer cells To address the effects of PL on cell viability, MTT assays were performed in L02, HUVECs, HeLa, MCF7 and MGC-803 cells. As shown in Fig. 3A, PL inhibited the viability of all three cancer cells in a dosedependent manner after 24 h and 48 h treatment. The IC50 values are 12.89 μM and 10.77 μM for HeLa cells, 13.39 μM and 11.08 μM for MCF-7 cells, and 12.55 μM and 9.725 μM for MGC-803 cells respectively. 15 μM of PL significantly suppressed the viability of cancer cells by 70-80% upon 48 h of treatment. In contrast, PL treatment had little effect on the cell viability of L02 cells and HUVECs. Colony formation assay further indicated that PL notably suppressed the growth of cancer cells (Fig. 3B). 12
Furthermore, we examined the levels of apoptosis by Annexin V/propidium iodide (PI) double staining assay in HeLa, MCF-7, and MGC-803 cells. These cells showed a high degree of staining after a 24 h treatment with PL, compared to the control (Fig. 3C). Based on Western blot analysis, both cleavedcaspase 9 and cleaved-caspase 3 levels were increased 24 h after PL treatment (Fig. 3D), suggesting that PL induces intrinsic apoptotic cell death.
3.4. FOXO3A knockdown rescues the piperlongumine-induced cell death To investigate whether the activation of the FOXO3A-BIM axis is directly involved in PL-induced cell death, we knocked down FOXO3A using a specific siRNA (siFOXO3A) in HeLa, MCF-7 or MGC-803 cells. As shown in Fig. 4A, siRNA-mediated knockdown of FOXO3A led to a significant decrease in FOXO3A protein levels, which effectively rescued the cells from PL-induced viability inhibition, compared to the control (Fig. 4B). Western blot analysis also showed that depletion of FOXO3A markedly attenuated the PL-induced activation of caspase 9, caspase 3 and BIM (Fig. 4C). These results indicate that PL induces cell death via the FOXO3A-BIM axis activation in cancer cells.
3.5. Piperlongumine inhibits xenograft tumor growth in nude mice To evaluate the in vivo impact of PL treatment, we used a subcutaneous xenograft model of MCF-7 cells in nude mice. When tumor sizes had grown to about 5-6 mm in diameter, PL was administered intraperitoneally (8 mg/kg) daily for two weeks. The results showed that PL treatment significantly reduced MCF-7 tumor volume and weight, compared to DMSO (vehicle)-treated controls (Fig. 5A-C). Mechanistically, immunohistochemical analysis revealed that PL treatment increased the abundance of 13
nuclear FOXO3A, and decreased the expression of phosphorylated FOXO3A (p-FOXO3A) protein in tumor tissues (Fig. 5D). Moreover, Western blot analyses revealed that PL enhanced the expression of BIM protein in MCF-7 tumors, compared to vehicle-treated controls (Fig. 5E). Taken together, these results indicate that PL inhibits tumor growth in vivo by activation of the FOXO3A-BIM axis.
3.6. Piperlongumine induces the dephosphorylation of FOXO3A by Akt signaling inhibition Because FOXO3A is critically regulated at the level of protein phosphorylation to regulate its subcellular localization and consequent transactivation activity, we investigated the level of p-FOXO3A following PL treatment in HeLa, MCF-7 and MGC-803 cells. Western blot analyses showed that the p-FOXO3A level was significantly decreased in response to PL treatment (Fig. 6A and B). As FOXO3A is a major effector of PI3K/Akt signaling [7], we next examined the role of PL in Akt activation. As shown in Fig. 6A and B, PL significantly suppressed Akt phosphorylation, without affecting its overall protein level. These data suggest that FOXO3A dephosphorylation and nuclear accumulation are mediated by Akt inactivation in response to PL (Fig. 6C).
4. Discussion Based on the results obtained in this study, we propose that the FOXO3A-BIM axis has a direct function in PL-induced cancer cell death: dephosphorylation and nuclear translocation of FOXO3A occurs as a result of Akt signaling inhibition, and the nuclear FOXO3A in turn specifically increases BIM expression to induce apoptosis in response to PL (Fig. 6C).
14
The FOXO family of transcription factors have emerged as an important target in cancer chemotherapy, because active FOXO members act as tumor suppressors by promoting cell cycle arrest and apoptosis [1, 6]. Within this family, activation of FOXO3A may either lead to cell cycle arrest by activation of the cell cycle inhibitor p27 Kip1 [12] and downregulation of cyclin D [27], or to apoptotic cell death by upregulation of pro-apoptotic proteins BIM and PUMA [13, 14]. It has been shown that downregulation of FOXO3A is associated with cancer progression [1], drug resistance and poor prognosis [2, 11, 28]. Thus, the past years have witnessed an increased attention to developing novel activators of FOXO3A as potential antitumor agents. Several anti-cancer drugs have been shown to increase the transcriptional activity of FOXO3A [26, 29, 30], which suggest it is a tangible therapeutic target for cancer therapy. In the current study, we found that the natural product PL induces nuclear retention of FOXO3A and also upregulates BIM expression, leading to apoptosis in a variety of human cancer cells, including breast, cervical and gastric cancer cell lines. Moreover, our data indicate that this pathway is also involved in the antitumor activity of PL in a xenograft mice model. These results suggest a promising application of PL as a potent FOXO3A activator, which might be expected to have potential preventive or therapeutic effects on the management of human cancer.
Amongst a large number of FOXO3A-dependent genes, there are several which are essential for initiation of the apoptotic program [31, 32]. In particular, FOXO3A is known to bind and activate the promoter of the pro-apoptotic gene BIM [26, 33]. Alternative splicing generates three BIM isoforms, including BimEL, BimL and BimS, which differ in their pro-apoptotic activity [34]. It is known that BIM proteins can provoke both the release of cytochrome C from mitochondria into cytosol and the activation of 15
caspases [34]. Thus, BIM up-regulation causes cellular apoptosis. In this study, PL treatment lead to FOXO3A binding to the BIM gene promoter, and up-regulation of BIM expression at both the mRNA and protein levels, which eventually resulted in cancer cell apoptosis by activation of caspase 9 and caspase 3. Importantly, it is well established that BIM has a crucial role in the anoikis of a variety of cancer cells, such as lung, breast, osteosarcoma and melanoma [35, 36]. The deficit of BIM protein leads to the occurrence of tumor metastasis and acquisition of chemotherapy resistance [34]. Therefore, BIM has attracted increasing attention as a plausible target for cancer therapy. Various chemotherapeutic agents use BIM as a mediating executioner of cell death (e.g. imatinib, gefitinib and bortezomib) [34]. Based on the effects of BIM on tumorigenesis and cancer treatment, our findings provide evidence that PL may be employed as a BIM-targeting agent for cancer therapies in the future, especially tumor metastasis and chemoresistance.
Since siRNA-mediated depletion of FOXO3A significantly attenuated PL-induced BIM expression and cell death, we concluded that FOXO3A-BIM axis is responsible for the observed apoptotic action of PL against cancer cells. However, the knockdown of FOXO3A in cells did not completely abolish PLmediated cell death. This result may reflect the fact that other mechanisms may be involved in the PLinduced cell apoptosis. Indeed, a previous study showed that PL treatment activates endoplasmic reticulum stress and mitochondrial dysfunction, and eventually induces apoptosis in gastric cancer cells [20]. These changes of apoptosis-related events could also contribute to PL lethality in cancer cells.
The transcriptional activity of FOXO3A is suppressed through the PI3K/Akt signaling pathway [37, 38]. 16
FOXO3A is phosphorylated by Akt at Thr32, Ser253 and Ser315, which results in its nuclear exclusion and cytoplasmic retention [12]. A hallmark of most cancers with a hyperactivated PI3K/Akt pathway (caused by RAS, PTEN or PI3K mutations) is the inactivation of FOXO transcription factors [39]. In contrast, PI3K or Akt depletion results in a significant activation of FOXO proteins, induction of apoptosis, decrease of cell viability and G1 cell cycle arrest [28]. In accord with these reports, our results show that PL treatment leads to an attenuation of Akt activity, resulting in nuclear retention of FOXO3A, which subsequently promotes cell cycle inhibition and induces a proapoptotic program in HeLa, MCF-7 and MGC-803 cell lines. Consistent with our results, a recent study revealed that PL capably reduces the activity of Akt/mTOR signaling through the generation of ROS [40]. Although PL-induced Akt inactivation is dependent on ROS accumulation, mechanisms underlying this biological process are not fully elucidated. In this respect, further studies are required to investigate how PL inhibits Akt signaling pathway in cancer cells. In addition, it has been reported that protein phosphatase 2A (PP2A) promotes the nuclear translocation and transcriptional activation of FOXO3A in response to PI3K/Akt inhibition through the dephosphorylation of Thr32 and Ser253 [41]. It will be interesting to explore whether the activity of PP2A is promoted in response to PL.
It is well documented that mitogen-activated protein kinase ERK phosphorylates FOXO3A at Ser425, degrading FOXO3A via the ubiquitin-proteasome pathway [15]. Research has shown that this phosphorylation event inhibits FOXO3A’s function to induce cell death and thus promote cell proliferation and tumorigenesis [15]. Interestingly, our results showed that PL also increase the level of pERK slightly (unpublished data). Although ERK is a negative regulator of FOXO3A-BIM axis, we 17
speculate that the activation of ERK may have evolved as another late feedback mechanism for eliminating PL’s cytotoxicity. More studies are needed to investigate whether the inhibition of ERK activation can sensitize cancer cells to PL treatment.
In summary, the findings presented in this report indicate that PL is capable of stimulating activation of FOXO3A-BIM apoptotic axis by inhibition of Akt signaling. Our findings not only provide important mechanistic insights into the oncostatic effects of PL, but also may lead to the development of a new strategy for the potential application of PL in cancer treatment.
Acknowledgements Funding was provided by the Scientific and Technological Research Program of Tianjin Municipal Education Commission (Grant No. 2017KJ007). We are grateful to Dr. Edward McKenzie (The University of Manchester, UK) for the critical reading of the manuscript. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
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FOOTNOTES The abbreviations used are: FOXO, forkhead box O; PL, piperlongumine; PI3K, phosphoinositide 3kinase; ERK, extracellular signal-regulated kinase; ROS, reactive oxygen species; HUVECs, human umbilical vein endothelial cells; PI, propidium iodide; p-FOXO3A, phosphorylated FOXO3A; p-Akt, phosphorylated Akt; HRP, horseradish peroxidase; IHC, immunohistochemistry
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Figure legends Figure 1. Effects of PL treatment on subcellular localization of FOXO3A. (A) Analysis of FOXO3A-GFP nuclear translocation. HeLa cells stably expressing FOXO3A-GFP were cultured in complete medium with or without 15 μM PL for the indicated time points. The fluorescent signals of FOXO3A-GFP were sequentially acquired by fluorescence microscopy. Cells treated with LY294002 (20 μM, 12 h) were used as a positive control. Hoechst staining (blue) was used to identify nuclei. Representative fluorescence images are presented. Scale bar, 20 μm. Comparable results were obtained in at least three independent experiments. (B) Effects of PL on nuclear localization of endogenous FOXO3A. HeLa, MCF-7 and MGC803 cells were treated with or without PL for the indicated time periods. Western blot analysis was used to monitor FOXO3A protein levels in the nuclear and cytoplasmic fractions. Right hand side panels indicate densitometric analysis of the intensities of Western blot by ImageJ software. FOXO3A values were normalized with nuclear marker Lamin C. Data are presented as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01). (C) Effects of PL on subcellular localization of FOXO3A in normal liver cell line L02. L02 cells stably expressing FOXO3A-GFP were treated with or without 15 μM PL for 24 h, and then fluorescent signals were acquired by fluorescence microscopy. (D) Same as (B) for detection of endogenous FOXO3A in L02 cells.
Figure 2. Effects of PL treatment on BIM expression. (A) The expression of BIM proteins after PL treatment. Total proteins were extracted from HeLa, MCF-7 and MGC-803 cells at the indicated time points following the treatment with 15 μM PL. The protein levels of BIM were detected by Western blot 22
using an antibody against BIM, β-actin was used as a loading control. The isoform BimEL expression was quantified by densitometric analysis and is represented as mean band intensity normalized to β-actin and related to untreated controls (Lower panels). (B) Effects of PL on BIM protein degradation. Cells were treated with PL for the indicated intervals in the presence or absence of 10 μM MG-132. Western blot was used to analyze the abundance of BIM protein. BimEL values were normalized with β-actin. (C) Effects of PL on BIM mRNA expression. The levels of BIM mRNA were examined in PL-treated HeLa or MCF-7 cells by real-time PCR, and GAPDH was used as loading control. (D) PL promoting FOXO3A binding to BIM promoter. ChIP analyses were performed in PL-treated L02 cells using a specific FOXO3A antibody or a control rabbit normal IgG. The PCR analyses of ChIP samples were performed using primers specific for BIM promoter fragment. Each ChIP sample was normalized to its corresponding input. All data are presented as means ± SD from three independent experiments (*P < 0.05, **P < 0.01).
Figure 3. PL treatment induces viability inhibition and apoptosis in cancer cells. (A) Effects of PL on the viability of normal and cancer cells. L02, HUVEC, HeLa, MCF-7, and MGC-803 cells were incubated with increasing doses of PL (2.5-15 μM) for 24 h or 48 h. Cell viability was determined by MTT assay. The normalized value of cell viability from the untreated cells was arbitrarily set as 1.0. The results from three biological replicates are presented as means ± SD. (B) HeLa, MCF-7, and MGC-803 cells incubated with PL (15 μM, 48 h) were used to determine the effect of PL treatment on cancer cell growth via colony formation assays. The efficiency of colony formation was quantified by ImageJ software. (C) Effects of PL on cancer cells apoptosis. HeLa, MCF-7, and MGC-803 cells were incubated with or without 15 μM PL for 24 h. The percentage of cell apoptosis was determined by Annexin-V/PI staining and flow 23
cytometry analysis. (D) Activation of caspase 9 and 3 in PL-treated cells. Cells treated with or without 15 μM PL for indicated intervals were subjected to Western blot analysis using antibodies against the cleaved-caspase 9 and cleaved-caspase 3. β-actin was used as a loading control. Data represent similar results from three independent experiments (*P < 0.05, **P < 0.01).
Figure 4. Effects of FOXO3A knockdown on PL activity. (A) siRNA-mediated knockdown of FOXO3A. HeLa, MCF-7 or MGC-803 cells were transfected with negative control siRNA (NC) or FOXO3A siRNA (siFOXO3A) for 24 h. Depletion of FOXO3A was determined by Western blot using an antibody against FOXO3A, and β-actin was used as a loading control. Densitometric analysis is represented as mean ± SD of three independent experiments. (B) Effects of FOXO3A knockdown on cell viability after PL treatment. Cells transfected with negative control or FOXO3A siRNA were treated with or without PL at the indicated concentrations for 48 h. Cell viability was analyzed by MTT assay. The results from three biological replicates are presented as means ± SD (*P < 0.05, **P < 0.01). (C) Effects of FOXO3A silencing on the expression of caspase 9, 3 cleavage and BIM. Cells were stimulated with or without 15 μM PL for indicated time points following transiently transfecting with negative control or FOXO3A siRNA. The corresponding changes in cleaved-caspase 9, cleaved-caspase 3 and BIM protein levels were measured by Western blot analysis. Data represent similar results from three independent experiments.
Figure 5. PL inhibits MCF-7 xenograft tumor growth in vivo, accompanied with increasing FOXO3A activity and BIM level. (A-C) PL treatment inhibited tumor volume and tumor weight of MCF-7 human breast cancer xenografts in nude mice. Tumors were measured twice weekly and their volumes were 24
calculated as π/6 × length × width. Tumors were removed and their weights were measured after two weeks. Data are represented as mean ± SD from six independent samples treated with vehicle or PL (**P < 0.01). (D) Immunohistochemistry to detect FOXO3A localization and p-FOXO3A level in tumor tissues. Representative images are shown from six tumor samples. Scale bar, 100 μm. (E) Expression of BIM protein in tumor tissues. The tumor tissues were lysed and protein was used to determine the BIM levels by Western blot analysis. β-actin was used as protein loading control. Three samples were randomly selected from respective tumor tissues.
Figure 6. PL treatment inhibits the phosphorylation of FOXO3A and Akt. (A) HeLa, MCF-7 or MGC-803 cells were treated with or without 15 μM PL for indicated intervals. Cell lysates were subjected to Western blot analysis using the corresponding antibodies as shown in the figure. β-actin was used as a loading control. (B) p-FOXO3A, p-Akt or total FOXO3A levels were quantified by densitometric analysis and normalized to total FOXO3A, total Akt or β-actin. Data are presented as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01). (C) Schematic illustration of the underlying mechanism of PL’s anticancer activity.
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S0006-2952(19)30050-4 https://doi.org/10.1016/j.bcp.2019.02.012 BCP 13418
To appear in:
Biochemical Pharmacology
Received Date: Accepted Date:
9 November 2018 8 February 2019
Please cite this article as: Z. Liu, Z. Shi, J. Lin, S. Zhao, M. Hao, J. Xu, Y. Li, Q. Zhao, L. Tao, A. Diao, Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIM-mediated apoptosis in cancer cells, Biochemical Pharmacology (2019), doi: https://doi.org/10.1016/j.bcp.2019.02.012
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Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIMmediated apoptosis in cancer cells
Zhenxing Liu, Zhichen Shi, Jieru Lin, Shuang Zhao, Min Hao, Junting Xu, Yuyin Li, Qing Zhao, Li Tao, Aipo Diao*
School of Biotechnology, Tianjin University of Science and Technology, Key Lab of Industrial Fermentation Microbiology of the Ministry of Education, State Key Laboratory of Food Nutrition and Safety, Tianjin 300457, China
* Corresponding author at: School of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China. Email address: [email protected] (Aipo Diao)
ABSTRACT The transcription factor forkhead box O 3A (FOXO3A) is a tumor suppressor that promotes cell cycle arrest and apoptosis. Piperlongumine (PL), a plant alkaloid, is known to selectively kill tumor cells while sparing normal cells. However, the mechanism of PL-induced cancer cell death is not fully understood. We report here that an association of FOXO3A with the pro-apoptotic protein BIM (also known as BCL2like 11, BCL2L11) has a direct and specific function in PL-induced cancer cell death. Using HeLa cells stably expressing a FOXO3A-GFP fusion protein and several other cancer cell lines, we found that PL treatment induces FOXO3A dephosphorylation and nuclear translocation and promotes its binding to the 1
BIM gene promoter, resulting in the up-regulation of BIM in the cancer cell lines. Accordingly, PL inhibited cell viability and caused intrinsic apoptosis in a FOXO3A-dependent manner. Of note, siRNAmediated FOXO3A knockdown rescued the cells from PL-induced cell death. In vivo, the PL treatment markedly inhibited xenograft tumor growth, and this inhibition was accompanied by the activation of the FOXO3A-BIM axis. Moreover, PL promoted FOXO3A dephosphorylation by inhibiting phosphorylation and activation of Akt, a kinase that phosphorylates FOXO3A. In summary, our findings indicate that PL activates the FOXO3A-BIM apoptotic axis by promoting dephosphorylation and nuclear translocation of FOXO3A via Akt signaling inhibition. These findings uncover a critical mechanism underlying the effects of PL on cancer cells.
Keywords: FOXO3A, BIM, Piperlongumine, Apoptosis, Cancer cells, Akt
1. Introduction The FOXO3A protein, a member of the forkhead box O (FOXO) transcription factors, plays a pivotal role in the control of various cellular processes, including cell proliferation, cell cycle, metabolism, apoptosis, autophagy, stress resistance and longevity [1-4]. As transcription factors, FOXOs regulate the expression of their target genes via nuclear translocation modulated by post-translational modifications, such as phosphorylation, ubiquitination and acetylation [5]. Growth and survival signals activate Akt, which phosphorylates FOXO3A on its conserved serine and threonine residues. FOXO3A phosphorylation by Akt impairs its DNA binding activity and promotes its interaction with the chaperone protein 14-3-3, sequestering it in the cytoplasm and rendering it inactive [6, 7]. Similarly, glycogen synthase kinase-3 beta 2
(GSK-3β) and Ikappa B kinase (IKK) have also been shown to inhibit FOXO3A activity by direct phosphorylation [8, 9]. In contrast, FOXO3A protein is activated and released from 14-3-3 under oxidative stress through Jun N-terminal kinase (JNK) signaling [2, 7]. In addition, FOXO3A can also be inhibited by the deacetylase sirtuin 1 (SIRT1) by facilitating its ubiquitination and proteasomal degradation [10].
It has been implicated that the FOXO family is associated with cancer development [1]. The deregulation of FOXO functions will cause uncontrolled cell proliferation and accumulation of DNA damage, which results in carcinogenesis. Active FOXO members act as tumor suppressors by promoting cell cycle arrest and apoptosis [6]. Survival analyses showed that low expression of FOXO3A is associated with a poor prognosis of patients with ovarian cancer [11]. Several studies suggest that activation of FOXO3A inhibits tumor growth and survival, including prostate and breast cancer cell lines and acute myeloid leukemia. The depletion of PI3K/Akt signaling causes significant activation of FOXO3A transcriptional activity, resulting in either cell cycle arrest by cyclin-dependent kinase inhibitor p27 Kip1 activation [12], or apoptotic cell death by FOXO3A-dependent protein PUMA or BIM expression [13, 14]. In addition, extracellular signal-regulated kinase (ERK) phosphorylates FOXO3A at Ser425, degrading FOXO3A via the ubiquitin-proteasome pathway, which inhibits its function to induce cell death and thus promotes cell proliferation and tumorigenesis [15]. Recent evidence has implicated that ΔNp63α protein is a direct FOXO3A transcriptional target, and FOXO3A-mediated expression of ΔNp63α inhibits oncogenic PI3K-, Ras-, and Her2-induced tumor metastasis [16]. Moreover, FOXO3A plays an important role in casein kinase 1α (CK1α)-mediated regulation of autophagy in RAS-driven cancers. CK1α phosphorylates 3
FOXO3A to promote its nuclear exclusion, decreasing expression of autophagy genes and limiting autophagic recycling of nutrients. In contrast, inhibition of CK1α activity stabilizes FOXO3A to elevate autophagic flux, which sensitizes oncogenic RAS-driven cancer cells to lysosomotropic agents via hyperaccumulation of ineffective autophagic vesicles [17]. Thus, the finding for compounds that evoke activation and nuclear translocation of FOXO3A is a promising therapeutic strategy for cancer treatment.
Using HeLa cells that stably express a FOXO3A-GFP reporter, we performed a small-molecule screen to discover compounds that can activate FOXO3A. One compound, piperlongumine, is a potent inducer of nuclear translocation of FOXO3A. Piperlongumine (PL), a plant alkaloid derived from the long pepper (Piper longum), exhibits numerous key biological activities [18]. It has been reported that PL could induce pancreatic cancer cell death by inducing intracellular reactive oxygen species (ROS) accumulation [19]. It has been suggested that direct inhibition of ROS-clearance proteins, such as glutathione S-transferase pi 1 (GSTP1) or Thioredoxin reductase 1 (TrxR1), may contribute to PL-induced ROS accumulation in cancer cells [20]. However, the cellular targets and downstream signaling pathways involving in PL stimulation have not been fully defined.
Here, we provide first evidence that PL induces FOXO3A dephosphorylation and nuclear translocation in various cancer cell lines, including cervical, breast and stomach cancer cells, subsequently promoting expression of the pro-apoptotic protein BIM and activation of apoptosis. In contrast, PL-induced cell death is effectively rescued by FOXO3A knockdown. These findings provide a molecular mechanism to the proapoptotic action of PL and implicate FOXO3A as a relevant mediator of the antitumor activity induced by 4
PL.
2. Materials and methods 2.1. Cell culture and reagents The human cervical cancer cell line HeLa, breast cancer cell line MCF-7, gastric cancer cell line MGC803, normal liver cell line L02 and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC). The cells were routinely cultured in Dulbecco’s modified Eagle’s Medium (Gibco, Eggenstein, Germany) containing 10% fetal bovine serum (Gibco), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO2 at 37°C. PL (purity: 99.33%) and LY294002 (99.84%) were purchased from Selleckchem (Houston, TX, USA). PL was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 15 mM. Complete media was used in all experiments, and the concentration of the vehicle, DMSO, was 0.1% in final incubations. Control cells were treated with vehicle alone. All chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Antibodies against FOXO3A (#12829), p-FOXO3A (#9466), BIM (#2933), cleaved-caspase 9 (#9501), cleaved-caspase 3 (#9664) and Lamin A/C (#4777) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phosphorylated Akt (p-Akt) (sc-514032), Akt (sc-56878) and β-actin(sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The horseradish peroxidase (HRP)-conjugated goat anti-rabbit (LK2001) or antimouse secondary antibodies (LK2003) were purchased from Sungene Biotech (Tianjin, China). FITC Annexin V apoptosis Detection Kit and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ, USA). 5
2.2. Plasmid construction and stable cell line The full length human FOXO3A gene (GenBank Accession Number NM_001455.3) was amplified by PCR using forward (5’-AACTCGAGATGGCAGAGGCACCGGCT-3’) and reverse (5’TTGGATCCTTGCCTGGCACCCAGCTCTG-3’) primers, and the cDNAs of HeLa cells as template. The PCR fragment of FOXO3A digested with XhoI and BamHI (New England Biolabs, Ipswich, MA, USA) was cloned into pLVX-AcGFP-N1 plasmid (Clontech, Palo Alto, CA, USA) to generate the FOXO3AGFP transfection vector. All constructs were verified by DNA sequencing. Lentivirus particles were generated by transfecting HEK-293T cells with pMD2.G and psPAX2 packaging plasmids and the corresponding backbone plasmid using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Virus were collected 48 h post transfection, filtered, and used fresh for infection overnight in the presence of 10 μg/mL polybrene. HeLa cell line stably expressing FOXO3AGFP was obtained by infection with pLVX-AcGFP-FOXO3A lentiviruses and selected in 1 μg/mL puromycin for at least two weeks.
2.3. Nuclear and cytoplasmic fractionation Subcellular fractionation of cells was performed using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, cells were homogenized in cytoplasmic extraction reagent I with subsequent addition of cytoplasmic extraction reagent II, and centrifuged. The supernatants from the centrifugation stage were cytoplasmic fractions. The insoluble pellets were suspended in nuclear extraction reagent, and nuclear proteins were extracted by 6
centrifugation. 2.4. Cell viability and colony formation assay Cells were seeded in 96-well plates at a density of 8 × 103 cells per well, and cell viability was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. After exposure of cells to PL, MTT solution (20 μL, 0.5 mg/mL in PBS) was added and incubated at 37°C for 4 h. Then, DMSO (200 μL/well) was added to dissolve the formazan dyes and the absorbance was measured at 570 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). For the colony formation assay, cells were plated (1500 cells/well) in 6-well plates and cultured for 7 days in growth medium. After PL treatment (15 μM, 48 h), the colonies were stained with crystal violet (0.2% w/v in 2% ethanol) and photographed under phase contrast microscope. The efficiency of colony formation was quantified by ImageJ software (Wayne Rasband, NIH, Bethesda, MD, USA).
2.5. Quantitative real-time PCR (qPCR) Total RNA was extracted from cells using a Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RNA was reverse-transcribed into cDNAs using Revert Aid First Strand cDNA Synthesis kit (Thermo Scientific). The qPCR was performed on an ABI Prism 7900HT Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using Fermentas Maxima SYBR Green/ROX qPCR reagents (Thermo Fisher Scientific). The reactions were carried out in a 96-well plate at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. GAPDH expression was used as an endogenous control to normalize target gene expression by the ΔΔCt method. Primers used were as follows: BIM, 5’ATGGCAAAGCAACCTTCTGA-3’ and 5’-CGCATATCTGCAGGTTCAGCC-3’; GAPDH, 5’7
CAAGGTCATCCATGACAACTTTG-3’ and 5’-GTCCACCACCCTGTTGCTGTAG-3’. 2.6. Cell apoptosis analysis Cells were plated on 60-mm dishes for 12 h, and then treated with PL (15 μM) for 24 h. Cells were then harvested, washed twice with ice-cold PBS, and evaluated for apoptosis by double staining with FITC conjugated Annexin V and PI in binding buffer for 30 min using a FACSCalibur flow cytometer (BD Biosciences, CA, USA).
2.7. ChIP assay ChIP assays were performed in HeLa cells with ChIP-IT Kit (Active Motif, Carlsbad, CA, USA) using an antibody specific for FOXO3A or normal rabbit IgG according to the manufacturer’s protocols. ChIP samples were subjected to PCR experiments to amplify fragment of the BIM promoter region using primers as follows: 5’-CGGCTTAGAAACTCAGGGCA-3’ (forward) and 5’CAGGCTCGGACAGGTAAAGG-3’ (reverse). To examine the strength of antibody binding to FOXO3A-sites, ChIP samples were subjected to qPCR using the same primers. The Cp value of each ChIP sample was normalized to its corresponding input.
2.8. RNA interference To silence FOXO3A gene expression, the following sequences were synthesized (RIBOBIO, Guangzhou, China): the FOXO3A mRNA target sequence 5’-GCACAGAGUUGGAUGAAGU-3’, the siRNA sense strand 5’-GCACAGAGUUGGAUGAAGUUU-3’, and the antisense strand 5’ACUUCAUCCAACUCUGUGCUU-3’. Transfection of siRNA duplexes was performed with 8
Lipofectamine 2000 reagent according to manufacturer guidelines (Invitrogen). Experimental assays were performed 24 h post-transfection. A non-target siRNA (Invitrogen) was used as a negative control.
2.9. Western blot Cell lysates were prepared by extracting proteins with RIPA buffer (Sigma) containing protease cocktail inhibitor (Roche, Basel, Switzerland). Equal amounts of protein samples were subjected to SDS-PAGE and transferred to a nitrocellulose membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST), the membrane was incubated with primary antibodies diluted in blocking solution (1:1,000) at 4˚C overnight. After washing with TBST, the membrane was incubated for an additional 1 h with the appropriate secondary antibodies conjugated to horseradish peroxidase at a dilution of 1:5,000. The protein bands were visualized using an enhanced chemiluminescence detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). The density of the immunoreactive bands was analyzed using ImageJ software.
2.10. Antitumor study in nude mice All animal experiments were approved by the Tianjin University of Science and Technology’s Policy on the Care and Use of Laboratory Animals. Five-week-old female BALB/c nude mice (18~20 g) purchased from Vital River Laboratories (Beijing, China) were used for in vivo experiments. MCF-7 cells were collected and injected subcutaneously (2 × 106 cells in 200 μL normal saline) into the right flank of nude mice. Six animals per group were used in each experiment. When tumor mass reached 5-6 mm in diameter (~7 days), mice were treated with PL at the dose of 8 mg/kg body weight (152 µg of PL in 200 μL normal 9
saline) by intraperitoneal (i.p.) injection once every other day of induction. Normal saline containing equal amount of DMSO was used as the vehicle control. Tumor volumes were measured twice weekly using a vernier caliper, and the volume was calculated according to the formula: π/6 × length × width of the tumor. At the end of experiment, the mice were sacrificed and the tumors were removed and weighed for use in the protein expression studies.
2.11. Immunohistochemistry (IHC) staining The xenograft tumors were fixed in formalin. For immunohistochemical staining, air-dried tissue sections were rinsed in PBS with 0.1% Triton X-100, then incubated with 0.3% hydrogen peroxide for 30 min to quench endogenous tissue peroxidases. After this, the sections were blocked with 10% goat serum for 30 min, incubated with the FOXO3A, p-FOXO3A antibodies or rabbit IgG overnight at 4°C, then labeled with goat anti-rabbit IgG conjugated to HRP. Immunostaining was visualized with diaminobenzidine (DAB) and stained with hematoxylin. Slides were dehydrated through alcohols and xylene and mounted in permount. Slides were photographed with a fluorescent microscope (Olympus BX53, Tokyo, Japan).
2.12. Statistical analysis All plots show individual data and means ± standard deviation (SD). Data were analyzed by unpaired Student’s t-test or one-way analysis of variance (ANOVA), using GraphPad Prism version 5.01 (GraphPad Software). In all cases, P < 0.05 was considered statistically significant.
3. Results 10
3.1. Piperlongumine treatment induces FOXO3A nuclear translocation in cancer cells Using HeLa cells stably expressing a FOXO3A-GFP fusion protein, we found that PL treatment potently induced FOXO3A-GFP nuclear translocation in a dose- and time-dependent manner. The spatio-temporal kinetics of FOXO3A nuclear translocation was analyzed by exposing the cells to 5, 10, 15 and 20 μM PL for 3, 6, 12 and 24 h (data not shown). The localization of this chimaeric protein was revealed by fluorescence microscopy. Significant nuclear accumulation of fluorescent signals were observed after treatment with 15 μM PL for 12 or 24 h, compared to the control (Fig. 1A). LY294002, a cell-permeable and broad-spectrum PI3K family inhibitor that acts on the ATP-binding site of the enzyme, has been shown to block PI3K-dependent Akt phosphorylation and kinase activity [21, 22]. The inhibition of PI3K/Akt signaling using LY294002 can cause FOXO3A translocation from the cytosol into the nucleus [23, 24]. In our study, similar results were also observed upon treatment with LY294002. To further confirm the nuclear translocation of endogenous FOXO3A in response to PL treatment, subcellular fractionation and Western blot analysis were performed in HeLa, MCF-7 and MGC-803 cells. Following treatment with 15 μM PL for 6, 12 and 24 h, nuclear FOXO3A abundance was significantly increased whereas cytoplasmic FOXO3A abundance was significantly decreased in these cell lines (Fig. 1B). Moreover, we examined the effect of PL on the subcellular localization of FOXO3A in normal liver cell line L02. The results showed that PL treatment did not induce FOXO3A nuclear translocation in L02 cells (Fig. 1C and D). These data indicate that PL induces FOXO3A nuclear translocation in cancer cells.
3.2. Piperlongumine treatment elicits upregulation of pro-apoptotic protein BIM To evaluate the role of PL in the transcriptional activity of FOXO3A, then the expression of FOXO3A 11
target genes were analyzed in HeLa, MCF-7 and MGC-803 cells. Western blot analysis demonstrated a strong increase in isoform BimEL protein levels, and detectable bands corresponding to BimL and BimS isoforms following 12, 24 h cell incubation with 15 μM PL (Fig. 2A). Moreover, research has shown that BIM proteins can be degraded via the ubiquitin-proteasome pathway [25]. MG-132, a proteasome inhibitor, dramatically increased the BimEL protein abundance in the presence of PL, compared to MG132 alone (Fig. 2B), suggesting that the BIM protein accumulation is not due to the inhibition of the ubiquitin-proteasome pathway. FOXO3A is known to bind and activate the BIM gene promoter [26]. Real-time PCR analysis revealed that BIM mRNA levels were significantly increased in HeLa and MCF-7 cells treated with 15 μM PL (Fig. 2C). In addition, data from ChIP experiments showed that the strength of FOXO3A binding to the BIM promoter was significantly increased in response to PL treatment, compared to the control (Fig. 2D). These results suggest that PL induces the expression of FOXO3A-BIM axis genes in cancer cells.
3.3. Piperlongumine inhibits cell viability and causes apoptosis in cancer cells To address the effects of PL on cell viability, MTT assays were performed in L02, HUVECs, HeLa, MCF7 and MGC-803 cells. As shown in Fig. 3A, PL inhibited the viability of all three cancer cells in a dosedependent manner after 24 h and 48 h treatment. The IC50 values are 12.89 μM and 10.77 μM for HeLa cells, 13.39 μM and 11.08 μM for MCF-7 cells, and 12.55 μM and 9.725 μM for MGC-803 cells respectively. 15 μM of PL significantly suppressed the viability of cancer cells by 70-80% upon 48 h of treatment. In contrast, PL treatment had little effect on the cell viability of L02 cells and HUVECs. Colony formation assay further indicated that PL notably suppressed the growth of cancer cells (Fig. 3B). 12
Furthermore, we examined the levels of apoptosis by Annexin V/propidium iodide (PI) double staining assay in HeLa, MCF-7, and MGC-803 cells. These cells showed a high degree of staining after a 24 h treatment with PL, compared to the control (Fig. 3C). Based on Western blot analysis, both cleavedcaspase 9 and cleaved-caspase 3 levels were increased 24 h after PL treatment (Fig. 3D), suggesting that PL induces intrinsic apoptotic cell death.
3.4. FOXO3A knockdown rescues the piperlongumine-induced cell death To investigate whether the activation of the FOXO3A-BIM axis is directly involved in PL-induced cell death, we knocked down FOXO3A using a specific siRNA (siFOXO3A) in HeLa, MCF-7 or MGC-803 cells. As shown in Fig. 4A, siRNA-mediated knockdown of FOXO3A led to a significant decrease in FOXO3A protein levels, which effectively rescued the cells from PL-induced viability inhibition, compared to the control (Fig. 4B). Western blot analysis also showed that depletion of FOXO3A markedly attenuated the PL-induced activation of caspase 9, caspase 3 and BIM (Fig. 4C). These results indicate that PL induces cell death via the FOXO3A-BIM axis activation in cancer cells.
3.5. Piperlongumine inhibits xenograft tumor growth in nude mice To evaluate the in vivo impact of PL treatment, we used a subcutaneous xenograft model of MCF-7 cells in nude mice. When tumor sizes had grown to about 5-6 mm in diameter, PL was administered intraperitoneally (8 mg/kg) daily for two weeks. The results showed that PL treatment significantly reduced MCF-7 tumor volume and weight, compared to DMSO (vehicle)-treated controls (Fig. 5A-C). Mechanistically, immunohistochemical analysis revealed that PL treatment increased the abundance of 13
nuclear FOXO3A, and decreased the expression of phosphorylated FOXO3A (p-FOXO3A) protein in tumor tissues (Fig. 5D). Moreover, Western blot analyses revealed that PL enhanced the expression of BIM protein in MCF-7 tumors, compared to vehicle-treated controls (Fig. 5E). Taken together, these results indicate that PL inhibits tumor growth in vivo by activation of the FOXO3A-BIM axis.
3.6. Piperlongumine induces the dephosphorylation of FOXO3A by Akt signaling inhibition Because FOXO3A is critically regulated at the level of protein phosphorylation to regulate its subcellular localization and consequent transactivation activity, we investigated the level of p-FOXO3A following PL treatment in HeLa, MCF-7 and MGC-803 cells. Western blot analyses showed that the p-FOXO3A level was significantly decreased in response to PL treatment (Fig. 6A and B). As FOXO3A is a major effector of PI3K/Akt signaling [7], we next examined the role of PL in Akt activation. As shown in Fig. 6A and B, PL significantly suppressed Akt phosphorylation, without affecting its overall protein level. These data suggest that FOXO3A dephosphorylation and nuclear accumulation are mediated by Akt inactivation in response to PL (Fig. 6C).
4. Discussion Based on the results obtained in this study, we propose that the FOXO3A-BIM axis has a direct function in PL-induced cancer cell death: dephosphorylation and nuclear translocation of FOXO3A occurs as a result of Akt signaling inhibition, and the nuclear FOXO3A in turn specifically increases BIM expression to induce apoptosis in response to PL (Fig. 6C).
14
The FOXO family of transcription factors have emerged as an important target in cancer chemotherapy, because active FOXO members act as tumor suppressors by promoting cell cycle arrest and apoptosis [1, 6]. Within this family, activation of FOXO3A may either lead to cell cycle arrest by activation of the cell cycle inhibitor p27 Kip1 [12] and downregulation of cyclin D [27], or to apoptotic cell death by upregulation of pro-apoptotic proteins BIM and PUMA [13, 14]. It has been shown that downregulation of FOXO3A is associated with cancer progression [1], drug resistance and poor prognosis [2, 11, 28]. Thus, the past years have witnessed an increased attention to developing novel activators of FOXO3A as potential antitumor agents. Several anti-cancer drugs have been shown to increase the transcriptional activity of FOXO3A [26, 29, 30], which suggest it is a tangible therapeutic target for cancer therapy. In the current study, we found that the natural product PL induces nuclear retention of FOXO3A and also upregulates BIM expression, leading to apoptosis in a variety of human cancer cells, including breast, cervical and gastric cancer cell lines. Moreover, our data indicate that this pathway is also involved in the antitumor activity of PL in a xenograft mice model. These results suggest a promising application of PL as a potent FOXO3A activator, which might be expected to have potential preventive or therapeutic effects on the management of human cancer.
Amongst a large number of FOXO3A-dependent genes, there are several which are essential for initiation of the apoptotic program [31, 32]. In particular, FOXO3A is known to bind and activate the promoter of the pro-apoptotic gene BIM [26, 33]. Alternative splicing generates three BIM isoforms, including BimEL, BimL and BimS, which differ in their pro-apoptotic activity [34]. It is known that BIM proteins can provoke both the release of cytochrome C from mitochondria into cytosol and the activation of 15
caspases [34]. Thus, BIM up-regulation causes cellular apoptosis. In this study, PL treatment lead to FOXO3A binding to the BIM gene promoter, and up-regulation of BIM expression at both the mRNA and protein levels, which eventually resulted in cancer cell apoptosis by activation of caspase 9 and caspase 3. Importantly, it is well established that BIM has a crucial role in the anoikis of a variety of cancer cells, such as lung, breast, osteosarcoma and melanoma [35, 36]. The deficit of BIM protein leads to the occurrence of tumor metastasis and acquisition of chemotherapy resistance [34]. Therefore, BIM has attracted increasing attention as a plausible target for cancer therapy. Various chemotherapeutic agents use BIM as a mediating executioner of cell death (e.g. imatinib, gefitinib and bortezomib) [34]. Based on the effects of BIM on tumorigenesis and cancer treatment, our findings provide evidence that PL may be employed as a BIM-targeting agent for cancer therapies in the future, especially tumor metastasis and chemoresistance.
Since siRNA-mediated depletion of FOXO3A significantly attenuated PL-induced BIM expression and cell death, we concluded that FOXO3A-BIM axis is responsible for the observed apoptotic action of PL against cancer cells. However, the knockdown of FOXO3A in cells did not completely abolish PLmediated cell death. This result may reflect the fact that other mechanisms may be involved in the PLinduced cell apoptosis. Indeed, a previous study showed that PL treatment activates endoplasmic reticulum stress and mitochondrial dysfunction, and eventually induces apoptosis in gastric cancer cells [20]. These changes of apoptosis-related events could also contribute to PL lethality in cancer cells.
The transcriptional activity of FOXO3A is suppressed through the PI3K/Akt signaling pathway [37, 38]. 16
FOXO3A is phosphorylated by Akt at Thr32, Ser253 and Ser315, which results in its nuclear exclusion and cytoplasmic retention [12]. A hallmark of most cancers with a hyperactivated PI3K/Akt pathway (caused by RAS, PTEN or PI3K mutations) is the inactivation of FOXO transcription factors [39]. In contrast, PI3K or Akt depletion results in a significant activation of FOXO proteins, induction of apoptosis, decrease of cell viability and G1 cell cycle arrest [28]. In accord with these reports, our results show that PL treatment leads to an attenuation of Akt activity, resulting in nuclear retention of FOXO3A, which subsequently promotes cell cycle inhibition and induces a proapoptotic program in HeLa, MCF-7 and MGC-803 cell lines. Consistent with our results, a recent study revealed that PL capably reduces the activity of Akt/mTOR signaling through the generation of ROS [40]. Although PL-induced Akt inactivation is dependent on ROS accumulation, mechanisms underlying this biological process are not fully elucidated. In this respect, further studies are required to investigate how PL inhibits Akt signaling pathway in cancer cells. In addition, it has been reported that protein phosphatase 2A (PP2A) promotes the nuclear translocation and transcriptional activation of FOXO3A in response to PI3K/Akt inhibition through the dephosphorylation of Thr32 and Ser253 [41]. It will be interesting to explore whether the activity of PP2A is promoted in response to PL.
It is well documented that mitogen-activated protein kinase ERK phosphorylates FOXO3A at Ser425, degrading FOXO3A via the ubiquitin-proteasome pathway [15]. Research has shown that this phosphorylation event inhibits FOXO3A’s function to induce cell death and thus promote cell proliferation and tumorigenesis [15]. Interestingly, our results showed that PL also increase the level of pERK slightly (unpublished data). Although ERK is a negative regulator of FOXO3A-BIM axis, we 17
speculate that the activation of ERK may have evolved as another late feedback mechanism for eliminating PL’s cytotoxicity. More studies are needed to investigate whether the inhibition of ERK activation can sensitize cancer cells to PL treatment.
In summary, the findings presented in this report indicate that PL is capable of stimulating activation of FOXO3A-BIM apoptotic axis by inhibition of Akt signaling. Our findings not only provide important mechanistic insights into the oncostatic effects of PL, but also may lead to the development of a new strategy for the potential application of PL in cancer treatment.
Acknowledgements Funding was provided by the Scientific and Technological Research Program of Tianjin Municipal Education Commission (Grant No. 2017KJ007). We are grateful to Dr. Edward McKenzie (The University of Manchester, UK) for the critical reading of the manuscript. Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article.
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FOOTNOTES The abbreviations used are: FOXO, forkhead box O; PL, piperlongumine; PI3K, phosphoinositide 3kinase; ERK, extracellular signal-regulated kinase; ROS, reactive oxygen species; HUVECs, human umbilical vein endothelial cells; PI, propidium iodide; p-FOXO3A, phosphorylated FOXO3A; p-Akt, phosphorylated Akt; HRP, horseradish peroxidase; IHC, immunohistochemistry
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Figure legends Figure 1. Effects of PL treatment on subcellular localization of FOXO3A. (A) Analysis of FOXO3A-GFP nuclear translocation. HeLa cells stably expressing FOXO3A-GFP were cultured in complete medium with or without 15 μM PL for the indicated time points. The fluorescent signals of FOXO3A-GFP were sequentially acquired by fluorescence microscopy. Cells treated with LY294002 (20 μM, 12 h) were used as a positive control. Hoechst staining (blue) was used to identify nuclei. Representative fluorescence images are presented. Scale bar, 20 μm. Comparable results were obtained in at least three independent experiments. (B) Effects of PL on nuclear localization of endogenous FOXO3A. HeLa, MCF-7 and MGC803 cells were treated with or without PL for the indicated time periods. Western blot analysis was used to monitor FOXO3A protein levels in the nuclear and cytoplasmic fractions. Right hand side panels indicate densitometric analysis of the intensities of Western blot by ImageJ software. FOXO3A values were normalized with nuclear marker Lamin C. Data are presented as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01). (C) Effects of PL on subcellular localization of FOXO3A in normal liver cell line L02. L02 cells stably expressing FOXO3A-GFP were treated with or without 15 μM PL for 24 h, and then fluorescent signals were acquired by fluorescence microscopy. (D) Same as (B) for detection of endogenous FOXO3A in L02 cells.
Figure 2. Effects of PL treatment on BIM expression. (A) The expression of BIM proteins after PL treatment. Total proteins were extracted from HeLa, MCF-7 and MGC-803 cells at the indicated time points following the treatment with 15 μM PL. The protein levels of BIM were detected by Western blot 22
using an antibody against BIM, β-actin was used as a loading control. The isoform BimEL expression was quantified by densitometric analysis and is represented as mean band intensity normalized to β-actin and related to untreated controls (Lower panels). (B) Effects of PL on BIM protein degradation. Cells were treated with PL for the indicated intervals in the presence or absence of 10 μM MG-132. Western blot was used to analyze the abundance of BIM protein. BimEL values were normalized with β-actin. (C) Effects of PL on BIM mRNA expression. The levels of BIM mRNA were examined in PL-treated HeLa or MCF-7 cells by real-time PCR, and GAPDH was used as loading control. (D) PL promoting FOXO3A binding to BIM promoter. ChIP analyses were performed in PL-treated L02 cells using a specific FOXO3A antibody or a control rabbit normal IgG. The PCR analyses of ChIP samples were performed using primers specific for BIM promoter fragment. Each ChIP sample was normalized to its corresponding input. All data are presented as means ± SD from three independent experiments (*P < 0.05, **P < 0.01).
Figure 3. PL treatment induces viability inhibition and apoptosis in cancer cells. (A) Effects of PL on the viability of normal and cancer cells. L02, HUVEC, HeLa, MCF-7, and MGC-803 cells were incubated with increasing doses of PL (2.5-15 μM) for 24 h or 48 h. Cell viability was determined by MTT assay. The normalized value of cell viability from the untreated cells was arbitrarily set as 1.0. The results from three biological replicates are presented as means ± SD. (B) HeLa, MCF-7, and MGC-803 cells incubated with PL (15 μM, 48 h) were used to determine the effect of PL treatment on cancer cell growth via colony formation assays. The efficiency of colony formation was quantified by ImageJ software. (C) Effects of PL on cancer cells apoptosis. HeLa, MCF-7, and MGC-803 cells were incubated with or without 15 μM PL for 24 h. The percentage of cell apoptosis was determined by Annexin-V/PI staining and flow 23
cytometry analysis. (D) Activation of caspase 9 and 3 in PL-treated cells. Cells treated with or without 15 μM PL for indicated intervals were subjected to Western blot analysis using antibodies against the cleaved-caspase 9 and cleaved-caspase 3. β-actin was used as a loading control. Data represent similar results from three independent experiments (*P < 0.05, **P < 0.01).
Figure 4. Effects of FOXO3A knockdown on PL activity. (A) siRNA-mediated knockdown of FOXO3A. HeLa, MCF-7 or MGC-803 cells were transfected with negative control siRNA (NC) or FOXO3A siRNA (siFOXO3A) for 24 h. Depletion of FOXO3A was determined by Western blot using an antibody against FOXO3A, and β-actin was used as a loading control. Densitometric analysis is represented as mean ± SD of three independent experiments. (B) Effects of FOXO3A knockdown on cell viability after PL treatment. Cells transfected with negative control or FOXO3A siRNA were treated with or without PL at the indicated concentrations for 48 h. Cell viability was analyzed by MTT assay. The results from three biological replicates are presented as means ± SD (*P < 0.05, **P < 0.01). (C) Effects of FOXO3A silencing on the expression of caspase 9, 3 cleavage and BIM. Cells were stimulated with or without 15 μM PL for indicated time points following transiently transfecting with negative control or FOXO3A siRNA. The corresponding changes in cleaved-caspase 9, cleaved-caspase 3 and BIM protein levels were measured by Western blot analysis. Data represent similar results from three independent experiments.
Figure 5. PL inhibits MCF-7 xenograft tumor growth in vivo, accompanied with increasing FOXO3A activity and BIM level. (A-C) PL treatment inhibited tumor volume and tumor weight of MCF-7 human breast cancer xenografts in nude mice. Tumors were measured twice weekly and their volumes were 24
calculated as π/6 × length × width. Tumors were removed and their weights were measured after two weeks. Data are represented as mean ± SD from six independent samples treated with vehicle or PL (**P < 0.01). (D) Immunohistochemistry to detect FOXO3A localization and p-FOXO3A level in tumor tissues. Representative images are shown from six tumor samples. Scale bar, 100 μm. (E) Expression of BIM protein in tumor tissues. The tumor tissues were lysed and protein was used to determine the BIM levels by Western blot analysis. β-actin was used as protein loading control. Three samples were randomly selected from respective tumor tissues.
Figure 6. PL treatment inhibits the phosphorylation of FOXO3A and Akt. (A) HeLa, MCF-7 or MGC-803 cells were treated with or without 15 μM PL for indicated intervals. Cell lysates were subjected to Western blot analysis using the corresponding antibodies as shown in the figure. β-actin was used as a loading control. (B) p-FOXO3A, p-Akt or total FOXO3A levels were quantified by densitometric analysis and normalized to total FOXO3A, total Akt or β-actin. Data are presented as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01). (C) Schematic illustration of the underlying mechanism of PL’s anticancer activity.
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