Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells

The International Journal of Biochemistry & Cell Biology 64 (2015) 126–135

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Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells Zheng Wang a,1 , Xingan Cheng a,b,∗,1 , Qianqian Meng a , Peidan Wang a , Benshui Shu a , Qiongbo Hu a , Meiying Hu a,∗ , Guohua Zhong a,∗ a Key Laboratory of Natural Pesticide and Chemical Biology, Ministry of Education, and Lab of Insect Toxicology, South China Agricultural University, No. 483, Wushan, Tianhe, Guangzhou 510642, People’s Republic of China b Institute of Natural Product Chemistry, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, People’s Republic of China

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Article history: Received 2 December 2014 Received in revised form 11 March 2015 Accepted 26 March 2015 Available online 4 April 2015 Keywords: Azadirachtin Lysosomal membrane permeabilization Cathepsins Caspase-3 Apoptosis Spodoptera frugiperda

a b s t r a c t Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Multiple regulatory factors associated with cell death were identified and proved to play important roles in apoptosis pathways. Recently, the lysosome has emerged as a key element in apoptosis pathways through selective lysosomal membrane permeabilization (LMP) and releasing proteases (Guicciardi et al., 2004; Kågedal et al., 2005). This apoptosis process can lead to several lysosomal changes, such as the increasing of lysosomal volume, secretion of proteases and total protease activity (Tardy et al., 2006), and changes in the subcellular localization of cathepsins B, D and L (Kirkegaard and Jäättelä, 2009). The cathepsin family of lysosomal proteases is an important hydrolase and is divided into

∗ Corresponding authors. Tel.: +86 20 85280308; fax: +86 20 85280308. E-mail addresses: anzai [email protected] (X. Cheng), [email protected] (M. Hu), [email protected] (G. Zhong). 1 These authors contributed equally to this paper. http://dx.doi.org/10.1016/j.biocel.2015.03.018 1357-2725/© 2015 Elsevier Ltd. All rights reserved.

cysteine, serine and aspartic cathepsins (Turk and Stoka, 2007; Turk et al., 2012). Especially, the cathepsins B, D and L are the most ubiquitous among the abundant lysosomal proteases (Rossi et al., 2004). In medical field, cathepsins are closely associated with many human diseases and their secretions could pose serious threats to human health. Many diseases, such as cancer, rheumatic arthritis, osteoarthritis, alzheimer, amyotrophy, multiple sclerosis and neuroblastoma, would occur with the secretion of cathepsins (Mort et al., 1984, 1998; Bever and Garver, 1995; Baici et al., 1995; Cataldo and Nixon, 1990; Sagulenko et al., 2008). In contrast to their tumorpromoting effects, there is also evidence to elucidate that they function as tumor suppressors (Berchem et al., 2002; Lopez-Otin and Matrisian, 2007; Hah et al., 2012; Hole et al., 2012; Marques et al., 2013). Regardless of the intensive studies done underlying the cathepsins, the role of cathepsins in cancer is still controversial and remained elusive. In general, activation of the caspase cascade after LMP would activate the intrinsic pathway. Cathepsin B, the main lysosomal protease of the brain parenchyma, is shown increasing effect on

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caspase-11 and -1 activity, which plays important roles in brain ischemia by promoting both apoptotic and inflammatory processes (Foghsgaard et al., 2001). Furthermore, cathepsin B contributed to apoptosis via caspase activation in Dengue virus infection (Morchang et al., 2013). Many studies of cathepsin L on human diseases revealed the cysteine cathepsin L could also be associated with apoptosis process. Quantitative immunohistochemical analysis of human carotid atherosclerotic lesions suggested that the expression of cathepsin L in symptomatic patients was increased more than those who were asymptomatic patients (Li et al., 2009), though its detailed mechanism is poorly understood. The fact that the cathepsins could be as an enhancer in apoptosis pathway triggered many attentions. Cathepsin D of the silkworm Bombyx mori (BmCatD) RNAi suggested that BmCatD is critically involved in the programmed cell death of the larval fat body and gut in silkworm metamorphosis (Gui et al., 2006). Moreover, many investigations showed that cathepsins, especially cathepsin B and L, were activated in the process of ovigenesis and caused the degradation of ootid (Matsumoto et al., 1997; Uchida et al., 2001, 2004). As such strategy underlying cathepsins-induced cell death could bring novel insight into control some important pests, such as Rhodnius prolixus and Ceratitis capitata (Ferreira-DaSilva et al., 2000; Rabossi et al., 2004). Thus, understanding the roles of cathepsins in apoptosis could provide a new prevention strategy for pest management. As a kind of botanical insecticide mainly extracted from the neem tree Azadirachta indica (A.Juss), azadirachtin (AZA) has attracted widely attention for pest control in the last few years (Isman et al., 1990; Schmutterer, 1990; Linton et al., 1997). Such semiochemical properties equipped strong antifeedant activities against many insect species, which is also companied by remarkable insect growth regulatory (IGR) activities and sterility effects (Mordue and Blackwell, 1993; Huang et al., 2004; Tan and Luo, 2011). Apoptosis can be induced by particular endogenous and exogenous factors (Arena et al., 1992). A great deal of research at the cellular level revealed that azadirachtin could affect cell proliferation and induce apoptosis (Salehzadeh et al., 2003; Anuradha et al., 2007; Kumar et al., 2007). Our previous studies showed that P53 protein was involved in cell cycle arrest, apoptosis induction and cell proliferation inhibition when the Spodoptera litura Sl-1 cells were treated with azadirachtin (Huang et al., 2011). Apart for that, the comprehensive understanding still requires more studies underlying other possible signal factors participating in AZA-induced apoptosis in insect cultured cells. Here, we analyzed the cell proliferation inhibition and morphological changes of lysosomes by using fluorescence microscope and flow cytometry methods during AZA-induced apoptosis. More importantly, we further proved the effects of cathepsin B (SCB) and cathepsin L (SCL) on Sf9 cells apoptosis exerted through the interferencing of activity inhibitors CA-074me (CA) and Z-FF-FMK (FF). Our data showed that lysosomal changes occurred in AZA-induced apoptosis and SCL activity had important repercussions in the sensitivity of Sf9 cells to AZA, which could have promotion implications.

2. Materials and methods 2.1. Cell line and culture conditions Spodoptera frugiperda Sf9 cells were cultivated at 27 ◦ C in 25 cm2 flasks in 3 mL Grace’s insect cell culture medium (Gibco, USA) containing 10% fetal bovine serum (FBS), 0.3% yeast extract, 0.3% lactalbumin hydrolysate and 0.3% peptone. Cells were seeded and planked with 0.5 × 106 –1 × 106 cells/mL density in 35 mm cultural plates for 24 h when the cells were at the optimum conditions for the following treatment.

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2.2. Evaluation of cells viability by 3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl (MTT) assays Cells in good condition were selected and incubated for 24 h at 27 ◦ C in the 96-well plates with 100 ␮L cell suspensions in each hole. Moderate quantity of AZA (the final concentration of 0.75 ␮g/mL dissolved in 0.1% dimethylsulforxide (DMSO)) was added into the cell suspensions. 0.1% DMSO was used as the control. After different treatment times (0, 12, 24, 36, 48 and 60 h, respectively), 10 ␮L freshly prepared MTT was added and the plates were incubated in the darkness for 4 h at 27 ◦ C. Then discarded the medium, and added 150 ␮L fresh DMSO to each well to dissolve the formazan crystal by orbital shaking in the darkness for 15 min. The absorbance was measured at 570 nm by a microplate reader (BioTek, USA). Cell viability was calculated by the equation as: Cell viability (%) = (ODtreatment /ODcontrol ) × 100%. 2.3. Morphological observation 2.3.1. Cell morphological observation Cells with the density of 0.5 × 106 –1 × 106 cells/mL were collected and incubated for 24 h in 12-well plates. Then cells at proliferation phase were treated with AZA for 0, 12, 24, 36, 48 and 60 h, respectively. Morphological characteristics of Sf9 cells were recorded with inverted phase contrast microscope (IPCM) (Olympus, Japan). Ultrastructure of cells were obtained by transmission electron microscope (TEM), the treated cells and control group were washed with 0.1 M PBS (phosphate buffer saline) three times before being fixed in 3% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M PBS buffer pH 7.2 for 2 h. After incubation time, they were rinsed with 0.1 M PBS and post-fixed in 1% osmium tetraoxide at room temperature for 1 h. All samples were washed three times with 0.1 M PBS following dehydration through an alcohol series and embedded in spur resin. The specimens were ultra sectioned to 60 nm thickness, stained with 0.5% uranyl acetate and lead citrate, and finally examined under TEM (TECNAIG2 12). 2.3.2. 4 ,6-Diamidino-2-phenylindole (DAPI) staining analysis Cells were seeded in 12-well plates, and exposed to 0.75 ␮g/mL AZA for 0, 12, 24, 36, 48 and 60 h, respectively, and then the nutrient supernatant was discarded. The uncovered cells were fixed in 4% paraformaldehyde for 15 min and washed with PBS twice. Finally, cells were stained in DAPI dye liquor (Southern Biotech Company, USA) with the final concentration of 1 mg/L for 15 min and washed in PBS once again. The samples were observed and photographed by fluorescence microscope (FM) (Olympus BX51, Olympus, Japan). 2.3.3. Lyso-Tracker Red probe staining analysis Cells were treated with AZA for different times (0, 12, 24, 36, 48 and 60 h, respectively), and incubated with 500 ␮L pre-incubated Lyso-Tracker Red fluorescent probe (diluted 1:15,000) at 27 ◦ C for 2 h, then removed the nutrient supernatant with fluorescent probe. The fresh nutrition solution was added and the lysosomes morphology changes were showed by FM (Olympus BX51, Olympus, Japan). For quantify the fluorescent value, the sample was mixed by successive aspiration with pipette for several times, then immediately transferred into black polystyrene 96-well plates. The relative fluorescence units of probe were read by fluorescence microplate reader (FMR) with excitation/emission wavelengths at 577 nm/590 nm (BioTek, USA). 2.3.4. Acridine orange (AO) staining analysis Cells were seeded in 6-well plates, and exposed to AZA for different times. Cells were then collected, and resuspended by PBS. Cell suspension were dyed by AO staining with the concentration of 1 ␮M and incubated for 15 min in the darkness. Cover slips were

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mounted over glass slides with cell suspension and immediately observed and photographed using FM (Olympus BX51, Olympus, Japan). 2.3.5. Annexin V-FITC/PI staining analysis Cells were seeded on cover slips before being cultured in 6well plates for 12 h, and then exposed to AZA for different times. Discarded the culture media, and washed cells with PBS twice. The purified cells were dyed by Annexin V-FITC and PI staining with proper concentration and incubated for 5–30 min in the darkness. The cover slips of cells were mounted over glass slides and immediately observed and photographed using FM (Olympus BX51, Olympus, Japan). 2.4. DNA ladder assay Genomic DNA of different treatment groups was isolated by genomic DNA extraction kit (Takara, Japan) and was subjected to agarose gel electrophoresis. 2.5. Apoptosis rate and cell cycle changes analysis Different treated cells were collected and washed with PBS twice. The purified cells were dyed by Annexin V-FITC and PI staining with proper concentration and incubated for 5–30 min in the darkness. Cell suspension was filtered with 400 mesh screens to remove the cell fragments. The samples were processed in a FACSCalibur (Becton Dickinson, USA) and data were analyzed using ModFit LT software (Becton Dickinson, USA). 2.6. Total RNA isolation Total RNA from different treatment cells was extracted using the total RNA isolation system kit (Biotech, China) according to the manufacturer’s instructions. The isolated RNA was reversely transcribed to first-strand cDNA with reverse transcriptase M-MLV (TaKaRa, China). Briefly, 3 ␮L of total RNA, 1 ␮L of dNTP Mixture (each 10 mM), 1 ␮L of Oligo (dT) primer (50 ␮M), and the addition of RNase free H2 O was added up to 10 ␮L, incubated at 65 ◦ C for 5 min and chilled in the ice for 3 min immediately. Then in same tube, 1 ␮L of RTase M-MLV (10 U/␮L), 2 ␮L of 5× Reverse Transcriptase buffer, 0.5 ␮L RNase Inhibitor (40 U/␮L) and the addition of RNase free H2 O was added with the final volume 20 ␮L. The reaction protocol was performed at 42 ◦ C for 60 min, 70 ◦ C for 15 min, and cooled with ice. The products were stored at −20 ◦ C. 2.7. Preparation of protein extracts 2.7.1. Total protein extraction Cells were exposed to AZA for different times. Both floating and attached cells were collected, washed with PBS and then centrifuged at 4000 rpm for 5 min. The supernatant was discarded and cells lysed in 200 ␮L proteoJHMTM cell lysis reagent (Fermentas/Thermo Fisher Scientific) supplemented with 2 ␮L protease and 2 ␮L EDTA. Cells were disrupted by ultrasonic wave, then cleared by centrifugation at 4000 rpm for 10 min. Total protein extracts were stored at −80 ◦ C 2.7.2. Cytosolic protein extraction Collected different treated cells by centrifugation at 3000 rpm for 5 min, and washed it with ice-cold PBS twice. Resuspended cells with 1.0 mL of cytosol extraction buffer mix (Sangon biotech, China) containing DTT (1 ␮L), protease inhibitors (1 ␮L) and phosphatase inhibitors (5 ␮L). Cells were homogenized in an ice-cold glass tissue grinder for 30–50 passes. Perform the task with the grinder on ice. Homogenate was transferred to an ice-cold microcentrifuge

Table 1 Primers used in this paper. Primers

Primer sequence

Primers for qRT-PCR 5 -GAAGTGAGGGACCAAGGAT-3 qSCBF qSCBR 5 -TCTGCGGAGAAGTGGAAAT-3 qSCLF 5 -CAGGGTGATGAGGAGAAGC-3 qSCLR 5 TCGGTGGACGAGCAGTT3 qCaspase-3F 5 -ATGAAGGCGACGCTCTG-3 qCaspase-3R 5 -CGAAATGCTCGTGGTTG-3 qGAPDHF 5 -GTGCCCAGCAGAACATCAT-3 qGAPDHR 5 -GGAACACGGAAAGCCATAC-3 Primers for RT-PCR RT-SCBF 5 -AGCATTTCGGTCTCGTCT-3 RT-SCBR 5 -ACATCAACAAGGAGTGGC-3 RT-SCLF 5 -TACGACAGCGAGGTGGAGG-3 RT-SCLR 5 -GATGGCGACGGAGATGGGT-3 RT-Caspase-3F 5 -GGCTCACCTTCTACTTCGT-3 RT-Caspase-3R 5 -CTGGGAGTGTATTTCCTTTT-3 RT-GAPDHF 5 -AAACTGTGGCGTGATGG-3 RT-GAPDHR 5 -GACCTGTTCCTCGGTGTAG-3 Primers for antibody preparation 5 -gtacatatgATGTCCCTGCTCGACCTCGTCCGG-3 Anti-SCLF Anti-SCLR 5 -gtagtcgacCTACACGAGGGGGTAGGAGGC-3

tube, and centrifuged at 4 ◦ C, 3000 rpm for 15 min after ice bath for 15 min. Transferred the supernatant to a fresh cold tube, and centrifuged at 4 ◦ C, 12,000 rpm for 30 min to remove lysosomes and mitochondria. Collected supernatant carefully, which was cytosolic protein extracts, and stored at −80 ◦ C. 2.8. Quantitative real-time PCR (qRT-PCR) and real-time PCR (RT-PCR) qRT-PCR reactions were performed with three technical replicates on BioRad iQ5 real-time PCR detection system using 200 ng of cDNA, 0.2 ␮M of primers and SYBR Premix Ex Taq (TaKaRa). The primers were designed with Primer 5.0 software and listed in Table 1. Amplification conditions consisted of an initial denaturation at 95 ◦ C for 30 s, followed by 40 cycles of 95 ◦ C for 10 s, 60 ◦ C for 30 s and 72 ◦ C for 20 s. The GAPDH gene was used as an internal gene. After the reaction was completed, analysis of the amplification and melting curves was performed according to the manufacturer’s instructions. The mean value was calculated using three independent biological samples, and then relative expression of different genes was analyzed according to the 2−Ct method (Livak and Schmittgen, 2001). The RT-PCR primers were also listed in Table 1. Amplifications were performed by denaturing at 94 ◦ C for 4 min, followed by 29 cycles (34 cycles for caspase-3) of 94 ◦ C for 1 min, 60 ◦ C (55 ◦ C for caspase-3) for 1 min and 72 ◦ C for 1 min, with a final extension at 72 ◦ C for 10 min. The amplification products were detected by 1.5% agarose gel electrophoresis and analyzed gel imaging and analysis system. 2.9. Expressions of recombinant and polyclonal antibodies preparation The cDNA fragment sequence of SCL gene was amplified with specific primer pairs (Table 1), which contain the restriction sites Nde I and Sal I, respectively. The PCR products with Nde I and Sal I was cloned into pET28a (Invitrogen, USA) with T4 DNA ligase (Takara, China) at 16 ◦ C, and then transformed to BL21 (DE3) competent cells (Takara, China). After the positive clone was identified, the single bacterial colony was then inoculated in liquid LB at 37 ◦ C until its A600 reached 0.8. IPTG (Isopropyl-d-thiogalactoside, 0.6 mM) was added and then incubated for 4 h at 28 ◦ C. The culture was harvested by centrifugation and lysed by the lysis solution (0.05 M PBS, 1 mM EDTA and 0.05 M NaCl, PH 7.0). The protein was

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purified by Ni-NTA column (Promaga, USA). Purified recombinant SCL protein was used to immunize rabbit as described previously (Cui and Xu, 2006). The serum of the immunized rabbit was collected as the polyclonal antibody. The serum titer was detected by an enzyme linked immunosorbent assay (ELISA) (Voller et al., 1980). And the antibody had no cross-reactivity. 2.10. Western blot Sf9 cells were exposed to 0.75 ␮g/mL AZA for different times. Both floating and attached cells were collected, and washed with PBS for three times. Cells were lysed in 0.2 mL RIPA Buffer (1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS to which inhibitors were added at time of use to the following final concentrations: PMSF, 1 mM; aprotinin, 0.2 trypsin inhibitor U/mL; sodium orthovanadate, 1 mM), sonicated, and stored at −20 ◦ C (Huang et al., 2011). Protein samples were separated by sodium dodecyl sulfate 12% polyacrylamide gel electrophoresis and electroblotted onto PVDF (polyvinylidene difluoride) membranes using tris–glycine transfer buffer on a mini-trans-blot electro-phoretic transfer tank (Bio-RAD, USA). Primary antibodies were anti-SCL and anti-caspase-3. Secondary antibody was HRP-conjugated antirabbit IgG. Finally chemiluminescence detection was performed using the ECL detection system (Bio-RAD, USA). 2.11. Statistical analysis Data are expressed as means ± S.E.M. of three independent experiments. One-way ANOVA was used to compare the effects of treatments. Statistical analyses were performed using SPSS 18.0 (SPSS, Inc., USA). The graphs were drawn with Origin 7.5 (Origin Lab, USA). 3. Results 3.1. AZA induces apoptosis and inhibits cell proliferation in Sf9 cells Sf9 cells were treated with 0.75 ␮g/mL AZA for 0, 12, 24, 36, 48 and 60 h, and then the cell viability was assessed by MTT test, respectively. The result showed that there was no significant difference in inhibition rate after 12 h treatment. But after treated for 24 h, the proliferation of cells has been inhibited significantly (Fig. 1A). The cell viability was 90.5%, 79.9%, 70.7% and 60.7% after treated for 24, 36, 48 and 60 h, respectively (Fig. 1A). Meanwhile, cellular morphological changes were observed under IPCM. Untreated-cells presented in round shape and adhered well (Fig. 1C). After treated with AZA, a small amount of apoptosis bodied and floating cells were observed initially in 12 h treatment group, and apoptosis bodies increased while adherent cells decreased with the extension of induction time (Fig. 1C). Ultrastructure of Sf9 cells in apoptosis induced by AZA was observed by TEM. In control group, the cells exhibited homogeneous cytoplasm, intact nucleus and a few lysosomes (Fig. 1D). However, after cells incubated in AZA, the type of lysosome became diversification gradually, such as from primary lysosome to secondary lysosome or residual body (Fig. 1D). Besides, nuclear changed with the passage of time, performing in chromatin condensation, nucleus shrinks and fragment (Fig. 1D). The nucleus morphological characteristic change of Sf9 cells was measured by staining with DAPI. The result showed that the control group still held all complete and circular nucleus while the treatment groups presented erose, swelled or paged nucleus (Fig. 1C). The cell apoptosis and cell circle of Sf9 in different treatment time points were detected sequentially. As shown in Fig. 1B, the apoptosis rates of treatment increased dramatically within 24 h

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compared with control group. And the maximum apoptosis rate appeared in 48 h, up to 38.06%. Analogously, cells with green and red fluorescence both increased after exposed to AZA, but untreated cells emitted a weak green fluorescence only (Fig. 1C), which indicated that the level of apoptosis might increased with increase in action time. AZA-treated group exhibited a high percentage of cells in G2/M phase as compared to the control group (Fig. 1E), which indicated a higher apoptotic level. Additionally, we immediately detected DNA fragmentation through agarose gel electrophoresis, and DNA ladder was observed clearly in treatment groups of 24 and 36 h (Fig. 1F). The results showed that AZA could induce cell circle arrest, inhibit cell proliferation and cause apoptosis in Sf9 cells. 3.2. AZA induces lysosomal changes and SCL release to the cytosol in Sf9 cells We next investigated the involvement of the lysosomal pathway in AZA-induced apoptosis in Sf9 cells. The changes of lysosomes in the process of apoptosis were measured by staining with the LysoTracker Red. Fig. 2A showed that the cells with red fluorescence increased after exposed to AZA as compared to the control group (low level of red fluorescence). Especially, after treatment for 24 h, intracellular fluorescent particles were increased and waxed significantly. The relative fluorescence units of treated-cells were higher than control group, and increased with time-dependent manner (Fig. 2B). Furthermore, the proportion of Annexin V-FITC positive cells has ballooned after treated with AZA (Fig. 2C). These results demonstrated that AZA-induced apoptosis in Sf9 cells was companied by the lysosomal changes in some respects, which may include differentiation of the type or quantity. And LMP was measured by staining with the lysosomotropic agent AO. AO is a weak base that moves freely across membranes when uncharged and accumulates in acidic compartments like lysosomes in its protonated form, where it forms aggregates that fluoresce bright red. LMP is associated with proton release, which renders lysosomes more alkaline and hence with decreased red fluorescence (Marques et al., 2013). FM analysis showed that a high level of red fluorescence and a low percentage green fluorescence in control cells (Fig. 2D), which indicated intact lysosome. By contrast, a decrease in the percentage of cells with red fluorescence was observed in the treated group (Fig. 2D). These findings indicated that AZA-induced apoptosis involves LMP. As lysosomal proteases are released from lysosomes to the cytosol after LMP, their detection in the cytosolic fraction is indicative of LMP. We therefore detected SCL in whole-cell extracts and in cytosolic fractions of control and treated cells by western blot. The result showed that SCL was expressed in whole-cell protein extracts and that treated cells exhibited higher levels of SCL than the control cells (Fig. 2E). Notably, we found that SCL levels decreased in whole-cell extracts when cells exposure to AZA for 24 and 36 h, but increased in the cytosolic fractions (Fig. 2E). The result indicates that AZA could induce SCL releasing to the cytosol and further suggesting that AZA induces a lysosomal pathway of apoptosis in Sf9 cells. 3.3. The effects of SCB and SCL on apoptosis induced by AZA in Sf9 cells To test whether the apoptotic phenotype of Sf9 cells exposed to AZA associated with SCB and SCL, SCB and SCL were inhibited with CA and FF respectively, and cells apoptosis inhibitor Z-VADFMK (VAD) was used as positive control. As shown in Fig. 3A, CA and FF inhibited more than 70% of the expression of SCB and SCL mRNA when cells were treated for 2 h. From Fig. 3B, CA-treated Sf9 cells incubated in 0.75 ␮g/mL AZA did not show significantly

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Fig. 1. Analysis of apoptosis and proliferation in sf9 cells treated with AZA. (A) Effects of AZA on cell proliferation. (B) Apoptotic rate of cells induced by AZA for different times. (C) Representative photographs of cell morphological change induced by AZA. IPCM: inverted phase contrast microscopy; DAPI and Annexin V-FITC/PI staining obtained by FM. (D) Representative photographs of ultrastructure of different treatment groups, obtained by TEM. (E) Analysis of cell-cycle phases distribution after treatment with AZA. (F) Agarose gel electrophoresis analysis of genomic DNA of cells treated by AZA. M: marker; 1–4: negative control, AZA treated for 12, 24 and 36 h, respectively. Note: Different letters in the same column indicate significant differences (p < 0.05, Duncan’s test). All data represent mean ± S.E.M. of three independent experiments.

alter at apoptotic level as compared to the negative control. By contrast, FF-treated Sf9 cells incubated in AZA exhibited a lower apoptotic rate than cells treated with AZA alone (Fig. 3B). However, the effect of inhibition is limited, the cells will enter apoptosis over time. For measuring the inhibitory effect of VAD, the same induction experiment was carried out. VAD-treated Sf9 cells did not exhibit apoptosis after exposed to AZA (Fig. 3B), but presented a phenomenon of cells death ultimately (not shown).

Moreover, the ultrastructure of Sf9 cells in apoptosis induced by AZA in the case of cells incubated with inhibitors was observed by TEM. Indeed, intracellular secondary lysosome increased and nucleus deformed significantly when Sf9 cells were treated with AZA for 24 and 36 h in the case of cells incubated with CA (Fig. 3D), but the cells had not occurred apoptosis obviously in the case of cells incubated with FF or VAD (Fig. 3D), which was in agreement with previous result that just SCL associated with AZA-induced

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Fig. 2. AZA induces lysosomal changes and SCL release to cytosol in sf9 cells. (A) Cells were incubated with AZA for different times: : untreated cells as negative control, -: cells treated for 6, 12, 24, 36 and 48 h, respectively. The changes of lysosome were detected by Lyso-Tracker Red and visualization by FM. (B) Relative fluorescence units of different treatments staining with Lyso-Tracker Red measured by FMR. (C) Annexin V-FITC staining with different treatments, and the proportion of staining cells detected by flow cytometry. (D) Representative images of AO staining obtained by FM. Cells were incubated with AZA for different times (above D). (E) Effect of AZA on the expression and release of SCL to the cytosol, contrast whole-cell extracts (Total) with cytosolic fractions (Cyto). GAPDH was used as a loading control. Note: Different letters in the same column indicate significant differences (p < 0.05, Duncan’s test). All data represent mean ± S.E.M. of three independent experiments.

apoptosis in Sf9 cells. It was further proved by DAPI fluorescent technique that cells presented some changes, such as uneven distribution and leakage of nucleoplasm, nucleus deformation, apoptotic body (Fig. 3D), after Sf9 cells were treated with AZA in the case of cells incubated with CA. However, when cells incubated with FF or VAD, such morphological features have not been observed (Fig. 3D). Lyso-Tracker Red staining demonstrated that inhibiting the activity of SCB by CA could not inhibit the increase of red fluorescence in the process of AZA-induced apoptosis in Sf9 cells (Fig. 3C). Conversely, the red fluorescence had been inhibited by FF and VAD (Fig. 3C). These results therefore indicated that SCL had a pro-apoptotic role in AZA-induced apoptotis in Sf9 cells, but not in the case of SCB. 3.4. The relationship between cathepsins (B and L) and caspase-3 in apoptosis induced by AZA in Sf9 cells To further clarify the function of SCB, SCL and caspase-3 in the process of AZA-induced apoptosis in Sf9 cells, the expression of SCB, SCL and caspase-3 mRNA and protein was calculated through RT-PCR, qRT-PCR and western blot assays. The results indicated that expression of SCL (Fig. 4A and C) and caspase-3 (Fig. 4B and D) mRNA increased significantly after cells induced by AZA comparing to control group, and the maximum of caspase-3 mRNA

appeared in 24 h (Fig. 4B and D). In addition, SCL mRNA (Fig. 4A and C) showed proportional changes with that of caspase-3 (Fig. 4B and D). In the case of cells incubated with FF, expression of caspase3 mRNA showed notable decrease within 36 h treatment of AZA (Fig. 5B and D), but subsequently showed gradual increase, which suggested that inhibitory effect of FF on the apoptosis of Sf9 cells induced by AZA were limited. Meanwhile, expression of SCL mRNA in treatment groups also presented significant decrease in the case of cells incubated with VAD (Fig. 5A and C). However, expression of SCB mRNA had no significant changes during apoptosis of Sf9 cells (Fig. 5A and C), indicating that SCB did not participate in the apoptosis of Sf9 cells induced by AZA. Accordingly, these results demonstrated that a SCL-dependent upstream signal pathway activated caspase-3-dependent downstream apoptotic signal pathway to participate in the apoptosis of Sf9 cells induced by AZA. Results from analysis of western blot assay indicated that expression of SCL (Fig. 6A) and pro-caspase-3 (Fig. 6B) proteins increased remarkably in the process of Sf9 cells apoptosis induced by AZA for 12 h, and pro-caspase-3 was cleaved into a cleavage product (cleavage-caspase-3) about 20 kD after cells treated for 24 h (Fig. 6B), indicating pro-caspase-3 was activated significantly and showed enhanced activity in time-dependent manner. VAD

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Fig. 3. Effects of inhibitors on apoptosis and proliferation in AZA-treated sf9 cells. (A) Inhibition effects of CA and FF on SCB and SCL. Cells were incubated with CA and FF for different times (B) Detection of apoptotic rate of cells induced by AZA for 36 h, in the case of cells incubated with inhibitors. (C) Effect of inhibitors on lysosomal changes in cells treated with AZA. -: cells were induced by AZA for 24 and 36 h respectively, in the case of cells incubated with CA (-), FF (-), and VAD (-). (D) Representative images of DAPI staining and ultrastructure of cells. Note: Different letters in the same column indicate significant differences (p < 0.05, Duncan’s test). All data represent mean ± S.E.M. of three independent experiments.

Fig. 4. Expression profile of SCB, SCL and caspase-3 genes in sf9 cells induced by AZA. The relative expression levels of SCB and SCL mRNA in different treatments were determined by qRT-PCR (A) and RT-PCR (C), 1–5: cells were treated by AZA for 0, 12, 24, 36 and 48 h, respectively. The relative expression levels of caspase-3 mRNA in different treatments were determined by qRT-PCR (B) and RT-PCR (D), 1–5: cells treated as in (C). Note: Different letters in the same column indicates significant differences (p < 0.05, Duncan’s test). All data represent mean ± S.E.M. of three independent experiments.

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Fig. 5. Effects of inhibitor on the expression of SCB, SCL and caspase-3 genes in AZA-induced Sf9 cells. Cells were induced by AZA for 24 and 36 h in the case of cells incubated with VAD for 2 h, and the relative expression of SCB and SCL mRNA in different treatments were determined by qRT-PCR (A) and RT-PCR (C), 1: negative control (untreated); 2: AZA-induced for 24 h. 3–5: AZA-induced for 12, 24 and 36 h in the case of cells incubated with inhibitor. (B) and (D): The relative expression of caspase-3 mRNA in different treatments in the case of cells incubated with CA, FF and VAD for 2 h, respectively, 1–5: cells were treated as (C) except inhibitor. Note: Different letters in the same column indicates significant differences (p < 0.05, Duncan’s test). All data represent mean ± S.E.M. of three independent experiments.

could decline the expression of SCL in translation level (Fig. 6C) and that inhibition of SCL activity by FF could cause caspase3 inactivation (Fig. 6D). These results further proved that SCL participated in apoptosis of Sf9 cells induced by AZA through caspase-3-dependent downstream apoptotic signal pathway in translation level. In addition, after Sf9 cells treated by AZA for 12 h, expression of caspase-3 mRNA reached to a higher level (Fig. 4B and D), but cleavage-caspase-3 was not detected (Fig. 6B), indicating that AZA activating caspase-3 to induce apoptosis of Sf9 cells showed time-dependent characteristics. That is, AZA firstly activated the genes of caspase-3 or other caspase family to promote

their expression in transcription or translation level, which then enhanced the amount of pro-caspase, and finally induced cells apoptosis by activating pro-caspase to form the mature caspase (e.g., cleavage-caspase-3). According to morphological analysis assay and mRNA expression analysis assay, it was further confirmed that caspase-3 was not the only executive protein in the apoptotic signal pathways. In addition, there might be other caspaseindependent pathways, such as the translocation of apoptosisinducing factor (AIF), to participate in the apoptosis of Sf9 cells induced by AZA besides caspase-dependent apoptotic signal pathways.

Fig. 6. Expression of SCL and caspase-3 in AZA-induced cells was determined by Western blot. (A) Expression of SCL in AZA-induced cells for different times. (B) Expression of caspase-3 in AZA-induced cells for different times. (C) Effect of VAD on expression of SCL in different treatments. (D) Effect of FF on expression of caspase-3 in different treatments. ␤-actin was used as a loading control.

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4. Discussion More and more investigations have indicated that lysosome is an important factor in programmed cell death of mammals, whose function includes not only digestion of apoptotic bodies but a series of changes in signal pathway, ultimately either apoptosis or necrosis (Boya et al., 2003; Cirman et al., 2004; Guicciardi et al., 2007). Relocation to the cytosol of lysosomal contents is of a moderate magnitude, a regulated and apoptotic type of degradation follows, while a massive release of lysosomal enzymes induces rapid and uncontrolled necrotic degradation without significant caspase activation (Brunk et al., 1997; Kågedal et al., 2001). By focusing our findings, the lysosomal membrane permeabilization and the release of lysosomal enzymes in AZA-induced apoptosis, we have been to explain the special functions of lysosome. In addition, cathepsins are often overexpressed in tumor cells and protected cells from pharmaceuticals-induced apoptosis (Palermo and Joyce, 2008; Marques et al., 2013). Meanwhile, there is also evidence that they function as tumor suppressors (Lopez-Otin and Matrisian, 2007). The released lysosomal enzymes induced by different external stimuli played different roles. We analyzed the action of SCB and SCL in the process of AZA-induced apoptosis, and found that the apoptosis levels did not change after inhibiting SCB with CA, but decreased markedly when SCL inhibited by FF. These results preliminarily showed that SCL played a role in promoting apoptosis induced by AZA in Sf9 cells. Previous study showed that cathepsin B and L were involved in apoptosis in mammalian cells. For example cathepsin B and activated pro-caspase-3 colocalized in the cytosol of apoptotic neurons of Wistar rats (Canu et al., 2005), and cathepsin L deficient cells (A549 human lung epithelial cells) showed increased sensitivity to apoptosis (Wille et al., 2005). These studies suggested that lysosomal protease may participated in apoptosis process in various cells, such as hepatocytes, neurocyte, tumor cells, and immune cells, through activating downstream targets (Roberts et al., 1997; Foghsgaard et al., 2001; Chwieralski et al., 2006). Analogously, cathepsins are implicated in a multitude of physiological and pathophysiological processes in insects. Lee et al. (2009) examined the expression profiles of B. mori cathepsins B (BmCatB) and D (BmCatD) during normal development and after RNA interference (RNAi)-mediated inhibition, and the results showed that BmCatB was associated with the programmed cell death of the fat body during metamorphosis, revealed that BmCatB and D contributed to B. mori metamorphosis. Another study on silk gland histolysis of B. mori during metamorphosis showed the involvement of cathepsin B- and L-like proteinases (Shiba et al., 2001). They also showed that cathepsin L correlated with larval moulting of Helicoverpa armigera (Liu et al., 2006). Despite numberous studies revealed that the cathepsins performed critical roles in regulating physical processes of insects, the detailed mechanism of the downstream signals and the resemblance with the mammals is still undefined. In this study, we aimed to investigate the mechanisms underlying AZA-induced apoptosis in Sf9 cells, focusing on the role of lysosome and cathepsin B and L. Our results demonstrated that inhibition of SCL enzymatic activity by FF could inhibit the activation of caspase3. That is, SCL is closely related to activation of caspase-3. However, the Sf9 cells will enter apoptosis as the extension of induction time even using FF, which revealed that SCL-dependent upstream signal pathway was not the only way to activate the caspase-3-dependent downstream apoptotic signal pathway. In summary, our research showed that AZA-induced apoptosis involve lysosomal changes (e.g., LMP) and activation of caspase-3 in Sf9 cells. Our findings also indicated that inhibition of SCL (not SCB) enzymatic activities can retard Sf9 cells going into AZA-induced apoptosis, suggesting that SCL might have a pro-apoptosis role in this process. And SCL, as cathepsins in mammal cells (Morchang

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