Background: Azadirachtin, one of the most promising botanical insecticides, has been widely used for pest control. Azadirachtin induces apoptosis in insect cell lines, including Sf9, SL-1 and BTI-Tn-5B1–4. Mitochondrial and lysosomal pathways are likely involved in the azadirachtin-induced apoptosis, however, detailed molecular mechanisms remain largely undefined. Results: Azadirachtin-induced apoptosis in Sf9 cells was verified by morphological observation, Hoechst 33258 staining, and a Caspase-3-based analysis. Comparative two-dimensional gel electrophoresis (2-DE) coupled with a linear ion trap quadrupole (LTQ)-MS/MS analysis identified 12 prominent, differentially expressed proteins following azadirachtin treatment. These differentially expressed genes are involved in regulating cytoskeleton development, signal transduction, gene transcription, and cellular metabolism. Knockdown gene expression of a gene encoding a DnaJ homolog enhanced apoptosis induced by azadirachtin in Sf9 cells. Conclusion: Azadirachtin treatment induces apoptosis in Sf9 cells and affects expression of multiple genes with functions in cytoskeleton development, signal transduction, gene regulation, and cellular metabolisms. Azadirachtin induces apoptosis at least partially by down-regulation of Sf-DnaJ in Sf9 cells. Keywords: Azadirachtin, Apoptosis, 2-DE, Sf-DnaJ1, RNAi Background Azadirachtin is also toxic to cultured insect cells. Azadirachtin, a prototypical botanical tetranortriter- Inhibition of cell proliferation has been observed in Sf9 penoid isolated from neem trees (Azadirachta cells derived from the ovaries of Spodoptera frugiperda, indica, A. Juss), is one of the most potent botanical che- SL-1 cells derived from Spodoptera litura, BTI-Tn-5B1–4 micals and has been used extensively in pest control [1– cells derived from Trichoplusia ni, and C6/36 cells derived 3]. Azadirachtin is effective against more than 550 species from Aedes albopictus [7–11]. Treatments of these cells of insect pests, including insects from Lepidoptera, Hem- with 10 to 100 nM azadirachtin result in completely inhib- iptera, Diptera and Orthoptera. The mode of action of ition of cell proliferation [7, 8]. Studies with some of the azadirachtin against insects include antifeedant effect and insect cell lines suggest that apoptosis is the cause of cell disruption of insect growth and development. On the death based on observed morphological, physiological, other hand, azadirachtin has little toxicity to mammals biochemical, and toxicological changes [9–12]. and decays fast in the environment [4–6], which makes it The high efficacy of azadirachtin against cultured cells a preferred choice for pest management in the field. and insects has attracted a great deal of attention to re- veal the molecular pathways for its mode of action. * Correspondence: firstname.lastname@example.org However, so far most information on molecular mecha- Benshui Shu and Jianwen Jia contributed equally to this work. nisms associated with azadirachtin toxicity has been Key Laboratory of Crop Integrated Pest Management in South China, Ministry obtained from cancer cell lines. Apoptotic signaling of Agriculture, Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, South China Agricultural University, Guangzhou, China pathways are activated in cancer cells following azadir- Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou achtin treatments, including the caspase-dependent 510642, China pathway, AIF-mediated pathway, p38 and JNK1/2 Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shu et al. BMC Genomics (2018) 19:413 Page 2 of 11 pathway, ROS-dependent MAPK pathway and death re- Biosciences (Uppsala, Sweden). Azadirachtin (95%) was ceptor pathway [13–15]. In insect cells, the p53 gene is obtained from Sigma (St.Louis, MO, USA). Other che- induced in azadirachtin-treated SL-1 cells, resulting in micals were domestic products with analytical grade. cell cycle arrest and the induction of apoptosis . Rabbit polyclonal antibodies against HSP40, TCTP and Using insect Sf9 cells, our group has previously GAPDH, respectively, were obtained from BOSTER demonstrated that both mitochondrial and lysosomal (Wuhan, China). pathways are involved in apoptosis after azadirachtin treatments [17, 18]. Specifically, we found that cathepsin Cell culture L released from lysosome to cytosol was induced in Sf9 cells were obtained from the State Key Laboratory azadirachtin-treated Sf9 cells, resulting in the activation for Biocontrol/Institute of Entomology, Sun Yat-Sen of caspase-3 . Despite significant progress has been University (Guangzhou, China), and were maintained at made, our knowledge on molecular components and 27 °C in 25 cm culture flasks (Corning, USA) contain- pathways leading to apoptosis in azadirachtin-treated ing 3 mL Hyclone SFX-insect cell culture medium sup- cells remains fragmented. plemented with 5% fetal bovine serum. The doubling Comparative proteomic analyses are powerful and ef- time under optimum conditions was 18–24 h and cells fective tools for large-scale identification of proteins in- were subcultured every 2 days. volved in a specific biological process. Two-dimensional gel electrophoresis (2-DE) combined with mass spec- Cell viability assay trometry (MS) has commonly used for proteomics and Sf9 cells were seeded onto a 96-well plate (5 × 10 /well) has been extensively applied to analyze the differentially and incubated for 24 h, then exposed to a series of con- expressed proteins in identical biological samples that centrations of azadirachtin for 24 h and 0.1% DMSO was are treated differently [19, 20]. For example, 10 proteins included as a control. Fifty μL of methylthiazoletetrazo- of S. litura (Fabricius) affected by azadirachtin signifi- lium (MTT) solution was added to each well and cells cantly have been identified using 2-DE, and six of them were incubated in dark for another 4 h. After removing are functionally assigned based on matrix-assisted laser the supernatant, 150 μL DMSO was added and mixed desorption/ionization time-of-flight (MALDI-TOF-MS) thoroughly with the pipette. Cell viability was measured . Two induced hemolymph proteins with functions based on absorbance at 490 nm using a microplate reader in lipid metabolism have also been identified using 2-DE (Thermo Scientific, Waltham, MA, USA). coupled with MS/MS from azadirachtin-treated Ostrinia furnacalis (Lepidoptera: Crambidae) . Twenty-one Morphological observation by inverted phase contrast differentially expressed proteins have been identified microscopy using the 2-DE/MS/MS method in azadirachtin-treated Cells seeded in 6-well plates were treated with 0.75 μg/mL Drosophila melanogaster larvae, with results indicating azadirachtin and 0.1% DMSO was used as control. Mor- that heat shock protein 23 is the potential target of aza- phological characteristics of cells at 0, 24, 48, and 72 h dirachtin action . after treatments were recorded by an inverted phase con- The objective of this study is to conduct a systematic trast microscope (Lecia, Japan), respectively. study on proteins expressed differentially in Sf9 cells after treatments with azadirachtin via comparative proteomic Hoechst 33258 analysis analyses. Twelve most differentially expressed proteins Hoechst 33258 is a blue fluorescent dye that could pene- were identified by linear ion trap quadrupole trate cell membranes and stain the cell nuclei with blue (LTQ)-MS/MS. Among these 12 proteins, Sf-DnaJ1 (DnaJ color. Sf9 cells treated with azadirachtin for 24, 36 and 48 h homolog to subfamily A member 1) was down-regulated were stained with 0.5 mL Hoechst 33258 solution for 5 min significantly in azadirachtin-treated Sf9 cells. Knockdown andthenwashedwithphosphate-bufferedsaline(PBS) of Sf-DnaJ1 via RNA interference (RNAi) resulted in twice for 3 min each time. The stained cells were observed increased apoptosis in Sf9 cells after azadirachtin treat- under the fluorescent microscope (Nikon, Japan). ments. Our results suggest that Sf-DnaJ1 is a regulator of azadirachtin-induced apoptosis. Analysis of caspase-3-like enzymatic activity Sf9 cells treated with azadirachtin were collected and Methods the caspase-3-like proteolytic activity was measured Chemicals using a Caspase-3 Colorimetric Assay Kit (KeyGEN Hyclone SFX-insect cell culture medium was purchased BioTECH, Nanjing, China). The cells were then washed from Thermo Scientific (USA), and fetal bovine serum with PBS twice and collected by centrifugation at (FBS) was purchased from Gibco (USA). IPG drystrip 2000 rpm for 5 min. Total cellular proteins were ex- and IPG buffer were purchased from Amersham tracted using a cold lysis buffer on ice for 20–60 min. Shu et al. BMC Genomics (2018) 19:413 Page 3 of 11 Protein concentrations were determined following the Protein digestion, LTQ-MS/MS and database searching Bradford approach . The solution containing 150 mg In order to locate protein spots with different intensity proteins with the Caspase-3 substrate (integrating spe- in gels, Coomassie Brilliant Blue G-250 was used to stain cific luminescence substrate) was incubated in dark at the gels for mass spectrometric analysis. The gels were 37 °C for 4 h. Caspase-3-like enzymatic activity was fixed with a buffer containing 40% ethanol and 10% measured based on the absorbance of samples measured glacial acetic acid for 1 h, washed with double distilled at 405 nm using a microplate reader (Thermo Scientific, water three times, stained with Coomassie Brilliant Blue USA). The Cacpase-3 inhibitor Z-VAD-FMK was also G-250 staining solution overnight, and decolorized in a used with the final concentration of 20 μM. destaining solution for at least 4 h. The significantly al- tered protein spots were located by comparing gels with control and treated samples side by side. The identified protein spots were cut out from the gel, further Preparation of protein samples destained with 30 mM potassium ferricyanide/100 mM The adherent Sf9 cells treated with 0.75 μg/mL azadir- sodium thiosulfate (1:1, v/v) for 20 min, and washed in achtin for 24 h and control group were washed with PBS Milli-Q water until the gels were completely destained. The twice and then mixed with 1 mL lysis buffer containing spots were kept in 0.2 M NH HCO for 20 min and then 40 mM Tris-base, 7 M urea, 2 M thiourea, 4% (w/v) 4 3 lyophilized. Each spot was digested in 12.5 ng/mL trypsin CHAPS, 2% (v/v) carrier ampholytes pH 3–10 and with 0.1 M NH HCO overnight. The peptides were ex- 65 mM DTT. The homogenates were shaken for 15 min 4 3 tracted with 50% Acetonitrile, and 0.1% TFA three times. in an ice-water bath and centrifuged at 14000 rpm for Separation and identification of the digested proteins 15 min at 4 °C. Protein concentrations of supernatants were conducted on a Finnigan LTQ mass spectrometer were determined by the Bradford method. (ThermoQuest, San Jose, CA, USA) coupled with a Surveyor HPLC system (ThermoQuest, San Jose, CA, 2-DE, gel staining and image analysis USA). A Microcore RP column (C18 0.15 mm × 120 mm; Before loading for 2-DE, samples were dissolved in ThermoHypersil, San Jose, CA, USA) was used to separate 350 μL rehydration buffer containing 7 M urea, 2 M the protein digests. Solvent A was 0.1% (v/v) formic acid, thiourea, 4% (w/v) CHAPS, 2% (v/v) IPG buffer, 20 mM and solvent B was 0.1% (v/v) formic acid in 100% (v/v) DTT, and a trace of bromophenol blue, then centrifuged ACN. The gradient was held at 2% solvent B for 15 min, at 14000 rpm for 5 min. Total protein extracts from and increased linearly to 98% solvent B for 90 min. The control and treated samples were separated through peptides were eluted from the C18 microcapillary column 2-DE. Two protein samples (140 μg) were loaded onto at a flow rate of 150 μL/min and then electrosprayed dir- analytical and preparative gels, respectively. Isoelectric ectly into an LCQ-Deca mass spectrometer with the appli- focusing (IEF) was carried out on an IPGphor system cation of spray voltage of 3.2 kV and capillary temperature (Amersham Biosciences) with pH 4–7 IPG strips (18 cm, at 200 °C. The full scan was ranged from M/Z 400 to linear) according to the manufacturer’s instructions. A 2000. Protein identification based on MS/MS data was total of 60 kVh was applied. Then the IPG strips were performed with SEQUEST software (University of equilibrated in 3 mL equilibration buffer twice for Washington, licensed to Thermo Finnigan) based on the 15 min. The first equilibration was performed in a buffer database of Swiss Port. The species for sequence search is containing 50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) Lepidoptera. Protein identification results were filtered with glycerol, 2% (w/v) SDS, 1% (w/v) DTT, and a trace amount a stringent filter condition of Xcorr (1 + ≥ 1.9, 2 + ≥ 2.2, of bromophenol blue. The second equilibration was per- 3+ ≥ 3.75) and DelCn (≥ 0.1). formed in a buffer modified by 2.5% w/v IAA instead of DTT. The strips were placed on the top of 12.5% Identification and sequencing of sf-DnaJ1 cDNA SDS-polyacrylamide gels and sealed with 0.5% agarose. To obtain a full length Sf-DnaJ1 cDNA, total RNA was Electrophoresis was carried out on a Hoefer SE 600 appar- isolated from Sf9 cells with an E.Z.N.A.™ Total RNA Kit atus (Amersham Biosciences) at 20 °C with the current of II (OMEGA, USA) according to the manufacturer’s in- 15 mA/gel for 40 min, and then 45 mA/gel for 6 h. The structions. First strand cDNAs were synthesized using a protein spots in gels were visualized by staining with silver PrimeScript® 1st Strand cDNA Synthesis Kit (TaKaRa) nitrate . At least three replicates were performed for according to the provided protocol. cDNA of Sf-DnaJ1 each sample. Images of each gel were acquired using was amplified by PCR with the degenerate primers Lab-Scan version 3.0 software (GEHealthcare) on an Sf-DnaJ1-F and Sf-DnaJ1-R (Additional file 1: Table S1) Image-Scanner. Images were analyzed by ImageMaster and 50 μL reaction mixture contained 0.5 μL template, 2-DE platinum version 5.0 software. The intensity of the 1 μL of each 10 mM primer, 0.5 μL Taq DNA polymer- protein spots was calculated with PDQuest 8.0 software. ase (TIANGEN, Beijing, China), 4 μL of 2.5 mM dNTP Shu et al. BMC Genomics (2018) 19:413 Page 4 of 11 mixture (TIANGEN, Beijing, China), and 5 μL 10× Taq DNA was amplified by PCR with the RNAi primers Buffer. The PCR program was performed with 32 cycles listed in Additional file 1: Table S1. The dsRNA against of 30 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C. The egfp, which used as the negative control, was similarly 5’-RACE (SMART RACE, Clontech) and 3’-RACE synthesized by the template pEGFP-C and primers in (TaKaRa) methods were used to fulfill the full-length Additional file 1: Table S1. The size and integrity of cDNA of Sf-DnaJ1. To ensure the 5′ and 3′ fragments dsRNAs were checked by agarose gel electrophoresis. were cloned from the same gene, specific primers were Transfection of Sf9 cells was performed based on the designed and PCR was used to amplify the coding region Lipofectin transfection method. Monolayer cultures of of the transcript encoding Sf-DnaJ1. Sf9 cells were prepared in 35-mm cell culture dishes (Corning, USA). Transfection was carried out by incuba- Quantitative real time PCR tion with 2 mL Hyclone SFX-insect cell culture medium In order to confirm the expression profiles of six identi- (without FBS) containing 5 μg dsRNAs overnight at 27 ° fied proteins, quantitative real-time PCR (qRT-PCR) was C, followed by incubation in 10 μL lipofectamine 2000 performed. Total RNA was extracted from Sf9 cells (Invitrogen) for 6 h. The medium was then replaced with treated with azadirachtin for 24 h and control cells using a medium containing FBS. After 24 h treatment, the a Total RNA Kit II (OMEGA, USA). The cDNA for RNAi efficiency was examined based on qRT-PCR and qRT-PCR was synthesized using a PrimeScript™ RT re- western blot results. agent Kit (TaKaRa, Japan), which has a gDNA Eraser to eliminate DNA contamination. qRT-PCR was performed Annexin V-FITC/propidium iodide double-staining and on CFX Connect™ Real-Time System (Bio-Rad, USA) flow cytometry using SsoAdvanced™ SYBR® Green Supermix (Bio-Rad, Anchorage-dependent Sf9 cells treated with 0.75 μg/mL USA). The PCR was carried out as follows: 95 °C for azadirachtin for 24 h were collected by centrifugation at 3 min for denaturation, 40 cycles of 95 °C for 10 s, 60 °C 2000 rpm for 5 min at 4 °C. The cells were then resus- for 10 s, 72 °C for 30 s, and a dissociation step at the pended and washed twice with PBS. The cells were then end. GAPDH was used as a reference for normalization. −ΔΔCT fixed in 500 μL binding buffer. Prior to cytometry ana- Relative expression levels were calculated by the 2 lysis, 5 μL Annexin V-FITC and 5 μL PI were added to method. Primers used in the experiments are listed in the fixed cells which were then incubated in dark for Additional file 1: Table S1. 15 min at room temperature. The cells were analyzed through a flow cytometry with an Ar laser with excita- Western blot assays tion and emission wavelengths 488 nm and 530 nm, re- Cells were collected and washed with PBS. Total cellular spectively. At least 2.0 × 10 cells were counted in each proteins were extracted using the CytoBuster™ Protein assay. The Sf9 cells with Sf-DnaJ1 knocked down and Extraction Reagent (Novagen, USA) according to the GFP control cells were treated with 0.75 μg/mL azadir- manufacturer’s protocol. Protein concentrations were de- achtin for 24 h and used for analyses. termined by the Bradford method. Equal amounts of pro- teins from different samples were separated on a 12% SDS-PAGE gel. Proteins in the gel were then transferred Data analysis to a polyvinylidene difluoride membrane (PVDF, Milli- Each treatment had three replicates and data were pore, USA). The membrane was washed with TBS for 3 expressed as the mean values ± SEM. One-way ANOVA times, incubated with TBS supplemented with 5% fat-free followed with Duncan’s new multiple range test (DMRT) milk at 4 °C overnight, and incubated with HSP 40 anti- and student’s t test were conducted during statistical body, TCTP antibody or GAPDH antibody at room analyses (P < 0.05). temperature for 2 h. Subsequently, the membrane was washed and incubated with the peroxidase-conjugated secondary antibody at room temperature for more than Results 2 h. The protein bands were detected by the enhanced Azadirachtin inhibited cell viability and proliferation chemiluminescence western blot kit (CW0049, CWBIO, As shown in Fig. 1a, the inhibition rates of Sf9 cell pro- Beijing, China) and detected by exposure to X-ray film a liferation were 14.6 ± 2.21%, 23.1 ± 3.32%, 28.7 ± 2.44%, dark room. 36.1 ± 1.56%, 43.8 ± 2.25% and 45.5 ± 1.35%, respectively, after cells treated with azadirachtin at the concentrations Double-stranded RNA synthesis and transfection of 2, 5, 10, 20, 40, 50 μg/mL for 24 h. These results re- dsRNA against the Sf-DnaJ1 transcript was synthesized vealed that azadirachtin had a strong inhibition effect on using a T7 RiboMAX™ Express RNAi System (Promega, cells proliferation and decreased cell viability in a USA) according to the provided protocol. Template dose-dependent manner. Shu et al. BMC Genomics (2018) 19:413 Page 5 of 11 Fig. 1 The analysis of proliferation inhibition and apoptosis induction by azadirachtin in Sf9 cells. a Cell viability of Sf9 cells after treated with multiple concentrations of azadirachtin for 24 h. b Morphological changes in Sf9 cells treated with 0.75 μg/mL azadirachtin at different times. c Cell nucleus morphology was detected by Hoechst 33258 staining. d The detection of Caspase-3 like activity in Sf9 cells after 0.75 μg/mL azadirachtin treatment for multiple time points. Different letters above bars indicate significant differences between different treatments by ANOVA followed by DMRT test (P <0.05) Morphological changes associated with apoptosis were stained homogeneous. In comparison, nuclei of Our previous study showed that a low concentration of azadirachtin-treated cells exhibited deeper blue staining azadirachtin induced apoptosis of Sf9 cells . As with nuclear and chromatin condensation after treat- shown in Fig. 1b, the morphological changes of Sf9 cells ment for 24, 36 and 48 h, respectively. The number of treated with 0.75 μg/mL azadirachtin could be observed live cells decreased gradually and nuclear condensation clearly under the inverted phase contrast microscopy. became more obvious with longer treatment time. Typical morphological characteristics of apoptosis were displayed in Sf9 cells treated with azadirachtin for 24 h, including cell shrinkage, increased gaps, membrane bleb- Azadirachtin increased caspase-3-like proteolytic activity bing and apoptotic bodies. After treatment for 48 h, gaps In order to examine if caspases were activated in Sf9 of cells increased and apoptotic bodies occurred widely. cells treated with azadirachtin, caspase-3-like proteolytic The number of viable cells and apoptotic bodies reduced activity was determined. Compared with that in control greatly after treatment for 72 h, and few cells kept the cells, the caspase-3-like proteolytic activity increased normal morphological appearance. These results suggest 2.66, 7.08, 9.26 and 9.91 fold in cells treated with azadir- that typical morphological characteristics of apoptosis achtin for 12, 24, 36 and 48 h, respectively (Fig. 1d). are induced in cells treated with 0.75 μg/mL azadirach- Apoptosis induced by azadirachtin was inhibited com- tin in Sf9 cells. pletely by Z-VAD-FMK and the caspase-3-like proteolytic activity in cells treated with both azadirachtin and Azadirachtin induced nuclear condensation Z-VAD-FMK was similar to normal cells (Additional file 2: As shown in Fig. 1c, Hoechst 33258 staining revealed Figure S1). These results indicate that azadirachtin in- nuclear changes induced by azadirachtin in Sf9 cells. duce apoptosis via activating caspase-3 activity in a The nuclei of control cells exhibited uniform sizes and time-dependent manner. Shu et al. BMC Genomics (2018) 19:413 Page 6 of 11 Identification of differential expressed proteins the 3′-untranslated regions. The predicted protein has Comparative proteomic analyses were performed to 404 amino acid residues with calculated molecular mass identify azadirachtin-responsive proteins. Approximately 45.46 kDa. The predicted Sf-DnaJ1 share 89, 77 and 67% 800 protein spots were detected on a 2-D gel using the sequence identity with DnaJ family proteins from Image Master 5.0 software. The criterion for proteins Bombyx mori, Tribolium castaneum and Aedes aegypti, with significant changes in abundance was at least a respectively. The phylogenetic relationship of Sf-DnaJ1 1.5-fold increase or decrease between control cells and with paralogs from other species is shown in Additional cells treated with azadirachtin for 24 h. Thirteen protein file 5: Figure S3. spots indicated by arrows satisfied this criterion and were considered differentially expressed proteins (Fig. 2). Validation of differentially expressed proteins by qRT-PCR Relative intensity of these proteins was showed in and western blot Additional file 3: Figure S2. Abundance of proteins cor- To examine the expression of the six genes affected by responding to spots D1 to D11 decreased in treated cells azadirachtin at transcriptional level, qRT-PCR was con- whilst abundance of proteins corresponding to spots U1 ducted. Consistent with the proteomic analysis, the levels and U2 increased in treated cells (Fig. 3). of the transcripts encoding Sf-DnaJ1, Sf-PS, Sf-P27BBP/ The identified azadirachtin-responsive proteins are eIF6, Sf-TCTP and Sf-PMSA6 decreased, whereas the listed in Table 1 and the matched peptide sequences level of the transcript encoding Sf-AWD increased in are listed in Additional file 4:Table S2.Proteins azadirachtin-treated Sf9 cells. Specifically, the level of up-regulated by azadirachtin were ribosomal protein L9 transcripts encoding Sf-DnaJ1, Sf-PS, Sf-P27BBP/eIF6, (Sf-RpL9, U1) and abnormal wing disc-like protein Sf-TCTP and Sf-PMSA6 decreased by 39.2, 43, 47, (Sf-AWD, U2). Proteins down-regulated were DnaJ 57.1and 23.7%, respectively (Fig. 4a), while the level of the homolog subfamily A member1 (Sf-DnaJ1, D1), ribosomal transcript encoding Sf-AWD increased 107.6%. Western protein SA (Sf-RpSA, D2), actin (D3-D5), beta-tubulin blot analysis revealed that DnaJ1 and TCTP decreased in (D6), proteasomezeta subunit (Sf-PS, D7), P27BBP/ Sf9 cells treated with azadirachtin for 24 h. eIF6-like (Sf-P27BBP/eIF6, D8), translationally-controlled tumor protein homolog (Sf-TCTP, D9), Proteasome sub- Knockdown of sf-DnaJ enhanced apoptosis induced by unit alpha type 6-A (Sf-PMSA6, D11), and an unknown azadirachtin protein (D10). To further confirm the role of Sf-DnaJ1 in azadirach- tin-induced apoptosis, dsRNA against Sf-DnaJ1 transcripts Characterization of sf-DnaJ1transcript was used for gene silencing. The transcript and protein A full length cDNA encoding Sf-DnaJ1 was cloned and levels of Sf-DnaJ1 were reduced significantly (98.2%) in sequenced as described in Materials and Methods. The dsRNA-treated cells compared to controls (Fig. 5a and b). nucleotide sequence was deposited in GenBank with the In addition, the cells treated with ds-DnaJ1 could not in- accession number KF562156. The Sf-DnaJ1-encoding duced apoptosis and the apoptotic rate had no significant cDNA is 1757 bp long with 148 bp at 5′- and 397 bp at difference with normal cells (Additional file 6:Figure S4). Fig. 2 The two-dimensional electrophoresis images of azadirachtin-treated Sf9 cells. a 2-DE gel of control sample. b 2-DE gel of the sample treated with 0.75 μg/mL azadirachtin for 24 h. Arrows indicate the differentially expressed protein spots, which were designated as D1–D11 and U1, U2. Among them, spots D1-D11 are down-regulated and spots U1and U2 are up-regulated Shu et al. BMC Genomics (2018) 19:413 Page 7 of 11 Fig. 3 The specific figures of different spots However, silencing the ds-DnaJ1-encoding gene resulted processes . Although it has been well documented in the enhancing of azadirachtin’s effect on apoptosis (Fig. that apoptosis is associated with azadirachtin treatments, 5c, d). the information on the molecules and pathways affected by azadirachtin remained fragmented. In order to iden- Discussion tify key regulators and uncover the molecular mechan- Azadirachtin has been proven to be an effective insecti- ism of apoptosis induced by azadirachtin, proteomic cidal ingredient for pest management and has the ability analyses including 2-DE coupled with MS-based protein to induce apoptosis in cultured cell lines . In this identification was carried out. Twelve proteins were study, Sf9 cells treated with 0.75 μg/mL azadirachtin ex- identified with significant changes after azadirachtin hibited morphological changes typical of apoptosis, as treatment (Fig. 2). have been previously observed in other insect cells in One of the down-regulated proteins was identified as vitro [11, 17]. Apoptosis is a form of physiological cell DnaJ homolog subfamily A member 1 (alternative name: death with important roles in various biological heat shock 40 kDa protein 4, hsp40–4). Heat shock Table 1 Identification of differentially expressed proteins in Sf9 cells treated with the azadirachtin by LTQ-MS/MS a) b) c) Spot no Protein name Fold change NCBI accession DP AAC MW PI Peptides identified number (%) (Da) D1 DnaJ homolog subfamily A member 1 1.61 ± 0.13 gi|14053203 2 2.00 45134.93 6.38 SGNDLILR D2 Ribosomal protein SA 2.85 ± 0.18 gi|54609281 3 7.84 33408.77 4.87 FAAHTGATPIAGR D3 Actin 8.97 ± 0.21 gi|46371991 6 13.70 40603.37 5.46 GYSFTTTAER D4 Actin 3.21 ± 0.11 gi|46371991 7 16.99 40603.37 5.46 GYSFTTTAER D5 Actin 1.92 ± 0.08 gi|46371991 7 16.99 40603.37 5.46 GYSFTTTAER D6 Beta-tubulin 10.43 ± 0.24 gi|74275413 3 10.14 33323.12 5.9 EVDEQMLNIQNK D7 Proteasome zeta subunit 6.26 ± 0.23 gi|114050993 7 36.63 26874.64 4.98 LFQVEYAIEAIK D8 P27BBP/eIF6-like 3.17 ± 0.14 gi|82880642 4 14.69 26304.74 4.63 VQFENNNEVGVFSK D9 Translationally-controlled tumor protein homolog 2.38 ± 0.16 gi|74837218 10 41.86 19938.81 4.67 LVETYAFGDKK D11 Proteasome subunit alpha type 6-A 1.73 ± 0.07 gi|114052160 3 13.41 27143 6.44 GTDAAVVAAQR U1 Ribosomal protein L9 5.06 ± 0.19 gi|112983495 6 25.79 21377.03 9.94 MAPGVTVVNSPK U2 Abnormal wing disc-like 2.80 ± 0.22 gi|153791847 5 27.92 17312.92 6.75 NIIHGSDSVESAK a) Fold change: D1-D11: the ratio of protein intensity of CK versus AZA; U1-U2: the ratio of protein intensity of AZA versus CK. b) Distinct peptides matched. c) Amino acids coverage. Shu et al. BMC Genomics (2018) 19:413 Page 8 of 11 Fig. 4 The expression level changes of differentially expressed proteins verified by qRT-PCR and western blot. a The mRNA expression profiles of six differentially expressed proteins in Sf9 cells with azadirachtin treatment for 24 h. Cells that treated with 0.1% DMSO were used as control. The GAPDH gene was used as the reference gene and data are expressed as arithmetic mean ± SEM of three independent experiments. Different letters above bars indicate significant differences between different treatments by ANOVA followed by student’s t test (P < 0.05). b the protein expression changes of DnaJ1 and TCTP in Sf9 cells after azadirachtin treatment for 24 h proteins (HSPs) have been recognized as common mo- significantly in Sf9 cells treated with azadirachtin, indi- lecular chaperones induced under various stress condi- cating that Sf-DnaJ1 was one of the regulators of azadir- tions, including heat shock, heavy metals, exposure to achtin action. Second, silencing Sf-DnaJ1-encoding gene radiation, ethanol, oxidative stress and so on . Previ- enhanced the capability of azadirachtin on inducing ous studies have provided substantial evidence that HSPs apoptosis. Our results indicated that Hsp40 can be an may regulate apoptosis. Hsp27 and Hsp70 have effects independent apoptosis regulator. Further studies are re- as anti-apoptotic proteins [29, 30]. Hsp60 and Hsp10 quired to reveal the molecular pathway between azadir- can enhance the proteolytic maturation of caspase-3 achtin and Sf-DnaJ1, and downstream steps. . Inhibition of Hsp70 and Hsp40 by HSP inhibitor Previous studies have shown that azadirachtin could in- KNK437 results in enhanced PS-341-induced cell death duce the depolymerization of actin, leading to cell arrest . Hsp40–Hsp70 pair plays a counter role through and subsequently apoptosis in a caspase-independent interacting with Bax, resulting in the inhibition of its manner [37, 38]. Our results provide further support to translocation to mitochondria in NO-induced apoptosis this observation. Compared with untreated control cells, . HSP 90 has been identified as a potential target of the abundance of actins (three protein spots correspond- azadirachtin effect through reversing docking . ing to D3, D4 and D5) and β-tubulin (D6) decreased dra- Hsp40 has been characterized as a co-chaperone in- matically in Sf9 cells treated with azadirachtin, suggesting volved in regulation of Hsp70 chaperone activity, but it that azadirachtin exerted its effects by inhibiting actin and remains unclear whether it can regulate apoptosis inde- tubulin to remodel apoptotic cells. pendently [35, 36]. In this study, we provided two pieces Proteasomes are ubiquitous and abundant multi-catalytic of evidence that, for the first time, demonstrated that a enzyme complexes in charge of the degradation of most Hsp40 member (Sf-DnaJ1) regulates apoptosis. First, we intracellular proteins . A previous study has showed found that the protein abundance of Sf-DnaJ1 decreased that a ubiquitin-proteasome complex is involved in the Shu et al. BMC Genomics (2018) 19:413 Page 9 of 11 Fig. 5 Silence of Sf-DnaJ1 enhanced apoptosis induced by azadirachtin. a The relative mRNA expression level changes of Sf-DnaJ1 in Sf9 cells with RNAi treatments. Control, normal cells; negative control, cells treated with unrelated dsRNA. b The protein expression levels of DnaJ1 in Sf9 cells with RNAi treatments. c The effects of RNAi treatments on apoptosis induced by azadirachtin in Sf9 cells were detected by flow cytometry. d The effects of RNAi treatments on apoptotic rate induced by azadirachtin in Sf9 cells. The data represent the mean values± S.E.M of three independent experiments. Different letters above bars indicate significant differences between different treatments at the same time (a represent p < 0.05) by ANOVA followed by DMRT apoptotic process . Proteasomes can stabilize study, the abundance of TCTP (D9) decreased in Sf9 pro-apoptotic proteins including p53 and Bax in various cells treated with azadirachtin, indicating that azadirach- cell types, but they also could suppress apoptosis induced tin could regulate the proliferation and apoptosis of Sf9 by some stimuli [41–44], suggesting that the roles of pro- cells via inhibiting the gene expression of TCTP. teasomes in apoptosis are complex and can go to either The abundance of three ribosome proteins was af- direction. In our results, the abundance of proteasome fected by azadirachtin. Azadirachtin reduced the abun- subunits zeta (D7) and alpha type 6-A (D11) decreased in dance of ribosomal protein SA (D2) and P27BBP/ Sf9 cells after azadirachtin treatment, indicating that aza- eIF6-like (D8), but elevated the abundance of the riboso- dirachtin could be a new proteasome inhibitor. mal protein L9 (U1). Ribosomal proteins are generally Translationally-controlled tumor protein homolog involved in protein synthesis. The impact of azadirachtin (TCTP) is generally regarded as a highly conserved pro- on the abundance of ribosomal proteins could suggest tein with multiple functions. It is widely expressed in that this chemical plays a role in protein biosynthesis. various tissues or cell types and plays an important role Several proteins including Bcl-2, NF-κB, P53 and PI3K in maintaining survival of a variety of cell types . have been previously suggested to be targets of azadirach- Several lines of evidence indicate that TCTP has a role tin [37–39]. However, these proteins were not identified in as an anti-apoptotic protein in cultured cells and it our proteomic analyses. One possible explanation is that protects cells from apoptotic cell death by inserting into the abundance of these regulatory proteins was less af- the mitochondrial membrane and inhibiting the fected by azadirachtin, and therefore, were not picked up dimerization of Bax . TCTP probably interacts se- in our analysis since we did focused on the proteins with lectively with actin and microtubule cytoskeleton to most differences. Alternatively, the pH range for our gel regulate cell shape during interphase and mitosis . strip was pH 4–7, which might not be optimal for separ- TCTP inhibits apoptosis by controlling the stability of ation of these proteins. More studies with different pH tumor suppressor p53, which in turns represses the tran- ranges should be used to identify other differentially af- scription of the TCTP-encoding gene [48, 49]. In this fected proteins after azadirachtin treatments. Shu et al. BMC Genomics (2018) 19:413 Page 10 of 11 Conclusion Abbreviations (LTQ)-MS/MS: Linear ion trap quadrupole-mass spectromtry; 2-DE: Two- In summary, the present study confirmed that azadirach- dimensional gel electrophoresis; AIF: Apoptosis inducing factor; DnaJ1: DnaJ tin induced apoptosis in Sf9 cells. Azadirachtin-responsive homolog subfamily A member1; JNK: c-Jun N-terminal kinase; ROS: Reactive proteins in Sf9 cells were analyzed and 12 differentially oxygen species; MAPK: Mitogen-activated protein kinase; MALDI-TOF- MS: Matrix-assisted laser desorption/ionization time-of-flight; FBS: Fetal expressed protein spots were identified through 2-DE bovine serum; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium and LTQ-MS/MS analyses. Transcription of the genes bromide; PI: Propidium iodide; IAA: Indole-3-acetic acid; IEF: Isoelectric encoding Sf-DnaJ1, Sf-PS, Sf-P27BBP/eIF6, Sf-TCTP, focusing; PVDF: Polyvinylidene difluoride; GAPDH: Glyceraldehyde-3- phosphate dehydrogenase; TCTP: Translationally-controlled tumor protein Sf-PMSA6 and Sf-AWD was also affected by azadirach- homolog; Sf-RpL9: Ribosomal protein L9; Sf-AWD: Abnormal wing disc-like tin. In addition, changes in protein levels of Sf-DnaJ1 protein Sf-RpSA: ribosomal protein SA; Sf-PS: Proteasomezeta subunit; Sf- and Sf-TCTP were confirmed by western blot. Knock- PMSA6: Proteasome subunit alpha type 6-A down of thegeneencodingSf-DnaJ1 byRNAiresulted Acknowledgements in increased apoptosis induced by azadirachtin, sug- The authors sincerely thank Prof. Ming-Shun Chen of Kansas State University gesting that Sf-DnaJ1 played an anti-apoptotic role in for revising this manuscript. azadirachtin-induced apoptosis of Sf9 cells. Overall, our Funding results indicated that azadirachtin triggered apoptosis by The work and publication costs were supported by grants from National down-regulating Sf-DnaJ1-encoding gene. Further experi- Nature Science Foundation of China (No. 31071713). ments are needed to obtain more evidence for a more de- Availability of data and materials tailed picture on the molecular processes of azadirachtin All data generated or analyzed during this study are available from this published toxicity on cultured cells and field insects. article and its Additional files. Authors’ contributions Conceived and designed the experiments: BS JJ GZ. Performed the experiments: Additional files BS JJ. Analysed the data: BS XY. Contributed reagents/materials/analysis tools: BS JJ JZ. Drafted the manuscript: BS. Revised the draft: SV XY GZ. All authors reviewed the manuscript. All authors read and approved the final manuscript. Additional file 1: Table S1. Details of the primer pairs used for genes cloning, RT-qPCR, RNAi. (DOCX 14 kb) Ethics approval and consent to participate Additional file 2: Figure S1. Z-VAD-FMK inhibited the apoptosis Not applicable. induced by azadirachtin in Sf9 cells. A: Morphological changes induced by different treatments in Sf9 cells. 1, 2, 3 and 4 were represented as normal Competing interests cells, cells treated with Z-VAD-FMK, cells treated with azadirachtin and cells The authors declare that they have no competing interests. treated with azadirachtin and Z-VAD-FMK, respectively. B: Caspase-3 like activity induced by different treatments in Sf9 cells. (TIF 3988 kb) Publisher’sNote Additional file 3: Figure S2. Quantitative analysis of the azadirachtin- Springer Nature remains neutral with regard to jurisdictional claims in published responsive proteins in Sf9 cells. The data are expressed as arithmetic maps and institutional affiliations. mean ± SEM of protein intensity on gels from three independent experiments. Statistical analysis was carried out using the SPSS software and different letters Author details above bars indicate significant differences between different treatments at the Key Laboratory of Crop Integrated Pest Management in South China, Ministry same time (P < 0.05) by ANOVA followed by DMRT. (TIF 792 kb) of Agriculture, Key Laboratory of Natural Pesticide and Chemical Biology, Additional file 4: Table S2. The matched peptide sequences of the Ministry of Education, South China Agricultural University, Guangzhou, China. identified proteins. (DOCX 14 kb) 2 Guangzhou City Key Laboratory of Subtropical Fruit Trees Outbreak Control, Additional file 5: Figure S3. The coding region sequence, deduced Zhongkai University of Agriculture and Engineering, Guangzhou, China. amino acid sequences and phylogenetic analysis of Sf-DnaJ1. 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Published: May 29, 2018