NCI-H446 cells were obtained from ATCC and stored under liquid nitrogen at the Prostate Diseases Prevention and Treatment Research Center, Jilin University, Changchun, China. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Biological Industries). Cells were grown in a humidified incubator maintained at 37°C and supplied with 5% CO₂. Cells were passaged by trypsinization (0.25% EDTA-free trypsin; Sangon Biotech Co., Ltd.) and reseeded in new culture dishes at one half of their original density. The day after cultures had reached a confluency of 70%, cells were transiently transfected with NC or SIRT3 expression constructs. RNA and protein were then extracted from cells 48 h post-transfection, for use in downstream experiments.

NCI-H446 cells were seeded in 96-well plates at a density of 6×10³ cells/well. After 24 h, the cells were transfected with either empty control plasmid (NC group) or plasmid encoding SIRT3 (SIRT3 group). Plasmids were constructed by Shanghai GeneChem Co., Ltd. Cells were then maintained under normal culture conditions until analysis of viability by MTT assay. To assess viability, 20 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sangon Biotech Co., Ltd.) was added per well of the 96-well plate. After a 4-hour incubation, 150 µl dimethyl sulfoxide (Sangon Biotech Co., Ltd.) was then added to each well. The absorbance of each well at 490 nm was then measured using a FLUOstar Omega microplate reader (BMG LABTECH). Cell viability was assessed at 24, 48 and 72 h post-transfection.

To prepare protein lysates for western blotting assays, cultured cells were washed with PBS, detached with a cell scraper and then lysed in 100–200 µl RIPA buffer (Beyotime Institute of Biotechnology) containing the protease inhibitor phenylmethylsulfonyl fluoride (Roche Diagnostics). Cell lysates were then sonicated on ice to disrupt protein aggregates by using ultrasonic disruptor UD-201 (TOMY SEIKO Co., Ltd.). Samples were then clarified by centrifugation at 15,000 × g for 20 min at 4°C, and the supernatants were retained. Clarified protein lysates (20 µg per lane) were then resolved by electrophoresis on 12% polyacrylamide gels and subsequently transferred onto polyvinylidene fluoride membranes (EMD Millipore). The membranes were blocked in Tris-buffered saline/0.5% Tween-20 (TBST) containing 5% non-fat milk for 1 h at room temperature, washed three times in TBST and then incubated with primary antibodies overnight at 4°C. The membranes were then washed three times in TBST before incubation with secondary antibodies for 1 h. After washing three times with TBST, the membranes were incubated with Hypersensitive ECL chemiluminescence reagent (Beyotime Institute of Biotechnology), and immunoreactivity was subsequently detected using a Syngene HR chemiluminescence imaging system (Synoptics). Primary antibodies, their respective dilutions and manufacturers, were as follows: anti-SirT3 (1:1,000; clone D22A3; product #5490), cleaved caspase-3 (1:1,000; product #9664), cleaved caspase-9 (1:1,000; product #9509), and BID (1:1,000; product #2002S) were purchased from Cell Signaling Technology, Inc.; anti-β-actin (1:1,000, cat. no. 66009-1-Ig) was obtained from Proteintech Group; anti-Bcl-2 (1:1,000, ab32124), anti-RIP (1:1,000; product code ab125072), RIP3 (1:1,000; product code ab152130), MLKL (1:1,000; product code ab183770), p-MLKL (1:1,000; product code ab196436), LC3 II (1:1,000; product code ab48394), and mutant p53 antibodies [Y5] (1:1,000; product code ab32049) were obtained from Abcam; anti-Mcl-1 (1:200; cat. no. sc-819), Bax (1:200; cat. no. sc-7480), and Ubiquitin (1:200; sc-8017) antibodies were purchased from Santa Cruz Biotechnology, Inc.; anti-Beclin-1 (1:1,000; cat. no. 612112) was obtained from BD Biosciences. Horseradish peroxidase goat anti-mouse (1:2,000; cat. no. SA00001-1) and goat anti-rabbit (1:2,000; cat. no. SA00001-2) antibodies were purchased from Proteintech Group.

Caspase-Glo 3/7 Assay (Promega Corporation) were used to determine cellular caspase-3/7 activities according to the manufacturer's instructions.

Total RNA was extracted from tissue samples and cell lines using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) based on standard protocols. NCI-H446 cells with SIRT3 overexpression treatment were cultured in a 6-well plate, washed twice with cold PBS and the culture medium was discarded. Then 500 µl of TRIzol per well was added and incubation followed for 5 min at room temperature. Chloroform, isopropanol and 75% ethanol were added successively, and mRNA was obtained by drying at room temperature after 7,500 × g centrifugation repeatedly. Subsequent generation of cDNA was performed using the EasyScript First-Strand cDNA Synthesis SuperMix kit according to the manufacturer's instructions (Beyotime Institute of Biotechnology). Briefly, in all reactions, 50–500 ng RNA was used as the starting template. The RT-PCR reaction was performed at 42°C for 30 min followed by an enzyme inactivation step at 85°C for 5 sec. Primer information and reaction conditions are presented in Tables I and II.

NCI-H446 cells were seeded in 6-well plates at a density of 3×10⁵ cells/well. After 24 h, the cells were transfected with either empty plasmid (NC group) or plasmid encoding SIRT3 (SIRT3 group). After 48 h, the cells were collected, washed twice with PBS, and apoptosis was then evaluated using the FITC Annexin V Apoptosis Detection kit (BD Biosciences). Briefly, cells were resuspended in 100 µl of 1X Binding Buffer before the addition of 5 µl Annexin V-FITC and 5 µl propidium iodide. The cells were then incubated in the dark for 15 min. An additional 400 µl of 1X Binding Buffer was added before analysis of apoptosis using a BD FACSCalibur flow cytometer (BD Biosciences).

Mitochondrial membrane potential was assessed using the Mitochondrial membrane potential assay kit with JC-1 (Beyotime Institute of Biotechnology), which accumulates in healthy mitochondria. NCI-H446 cells were seeded into 6-well plates at a density of 3×10⁵ cells/well. After experimental treatments, cells were harvested by digestion with EDTA-free trypsin. The cells were then resuspended in 1 ml serum-free DMEM containing 1 µl JC-1 (5 µg/ml) and incubated at 37°C for 20 min. At 3–5 min intervals, the cells were removed from the incubator and mixed by inversion to ensure uniform uptake of the dye. The cells were then pelleted by centrifugation at 1,500–2,000 × g for 5 min, washed with PBS and then resuspended in 400 µl DMEM. The fluorescence intensity of dye-labelled cells was analyzed using a BD FACSCalibur flow cytometer.

NCI-H446 cells were divided into five groups as follows; untransfected cells (CON group), cells transfected with empty plasmid (NC group), cells transfected with SIRT3 plasmid (SIRT3 group), cells treated with MG132 only (MG132 group), and cells transfected with SIRT3 plasmid and subsequently treated with MG132 (MG132+SIRT3 group). NCI-H446 cells were seeded into 6-well plates and 24 h later the appropriate groups were transfected with their respective plasmids and treated with MG132 inhibitor as indicated. After an additional 24 h, the cells were collected and lysates were prepared for analysis of protein expression by western blotting.

Cycloheximide (CHX) was used to evaluate changes in mutant p53 protein half-life. NCI-H446 cells were transiently transfected with a plasmid encoding SIRT3 48 h prior to CHX experiments. Cells were then treated with 50 µg/ml CHX, and cell lysates were subsequently obtained at 0, 0.5, 1, and 2-h time-points for analysis of protein expression by western blotting.

After NCI-H446 cells had been subjected to the indicated treatments, they were washed twice with PBS and lysed in RIPA buffer. Cell lysates were incubated for 30 min on ice with rotation. Lysates were then clarified by centrifugation at 14,000 × g for 15 min at 4°C, and the supernatants were subsequently retained and transferred to a fresh tube. Approximately 30–50 µl of clarified lysate was retained for analysis of input. Samples containing 1 mg total protein were incubated with 2 µg antibody overnight on ice. The following day, protein A/G-conjugated beads were added and the samples were then incubated for an additional night on ice. On the third day, the beads were pelleted by centrifugation for 5 min at 1,000 × g at 4°C and the supernatants were discarded. The beads were then washed 3–5 times with PBS. Finally, 60 µl RIPA buffer was added to 20 µl of bead slurry and 20 µl of sample was used/well for subsequent polyacrylamide gel electrophoresis.

Salmonella typhimurium A1-R is auxotrophic for arg and leu, which enables improved targeting and toxicity towards tumors, and this strain has previously been used in cancer treatment (12). Mouse-attenuated salmonella typhimurium phoP/phoQ was used in this study to deliver expression constructs to tumor cells in vivo. Attenuated salmonella typhimurium was induced into competence. Competent cells (100 µl) were mixed with 1 µl SIRT3 plasmid, and then transferred to a cold MicroPulser electroporation cuvette (0.1-cm gap). The cuvette was placed in a Gene Pulser Transfection Apparatus (product no. 1652100; Bio-Rad Laboratories, Inc.), and pulsed once. After hearing the beep, 1 ml LB liquid medium was immediately added into the cuvette. After gently resuspending the cells, they were transferred to a new centrifuge tube, cultured at 37°C for 1 h, and shaked at 220 RPM. Then the cells were plated on LB plates with ampicillin at 37°C for 12 h. The following parameters were used for electroporation: 2.5 kV, 25 µF, 200 Ω, 5 msec.

The animal study was approved by the Animal Ethics Review Committee of Basic Medical Sciences, Jilin University, in accordance with the regulations of the Institutional Committee for the Care and Use of Laboratory Animals of the Experimental Animal Center of Jilin University.

A total of 12 male 4 to 6-week-old BALB/C nu/nu nude mice weighing between 18–20 g were used for in vivo experiments. Animals were purchased from Huafukang Bioscience Co., Inc. Each mouse was injected subcutaneously with 2×10⁶ cells in a final suspension volume of 100 µl. Tumor size was then monitored daily by Vernier caliper until they had reached approximately 5 mm in diameter. Ten tumor-bearing mice were then randomly divided into two groups; the PQ group (animals injected with attenuated Salmonella transformed with empty plasmid) and the PQ-SIRT3 group (animals injected with attenuated Salmonella transformed with SIRT3 plasmid). Mice were intraperitoneally injected every 14 days with 5×10⁷ cfu of PQ or PQ-SIRT3-harboring bacteria. After treatment, the behavior and changes in water intake and weight of the animals were monitored. The maximum length (L) and short diameter (W) of the tumor was measured by Vernier caliper to calculate the tumor volume using the formula V=0.52 × L × W². The tumor growth index was used to record and plot tumor growth and was calculated using the formula: Tumor growth index=(V_n-V₁)/V₁ (where V₁ is the tumor volume of the first day, and V_n is the tumor volume of the nth day).

After 25 days of treatment, the nude mice were euthanized by cervical dislocation after intraperitoneal injection of 20% urethane (0.1 ml per mouse) and the tumors were removed, weighed and photographed. A small portion of each tumor was fixed in 10% formaldehyde solution for subsequent hematoxylin and eosin (H&E) staining. The remaining tumors were stored in liquid nitrogen for subsequent RNA and protein extraction.

The tumors were fixed in 10% formaldehyde solution for 48 h, and then embedded in paraffin. Histological sections were prepared by standard conventional processing and stained with H&E. The sections (0.2 mm) were incubated with 2% bovine serum albumin (BSA) for 30 min at RT to block nonspecifc binding, and then incubated with rabbit polyclonal anti-SIRT3 (1:200; product no. 2627; Cell Signaling Technology, Inc.) and anti-mutant p53 antibodies [Y5] (1:1,000; product no. ab32049), followed by incubation with goat anti-rat immunoglobulin G (IgG; 1:400; cat. no. SA00001-15; ProteinTech Group, Inc.) for 1 h at RT. Between each incubation step, the sections were washed 3 times with Tris-buffered saline with Tween-20 (12.5 mM Tris/HCl, pH 7.6, 137 mM NaCl and 0.1% Tween-20).

All the data were obtained after at least three independent experiments. Data are presented as the mean ± standard deviation. A Student's t-test was used to compare two groups and ANOVA with Tukey post hoc test was used to enable multiple comparisons between groups. P<0.05 was considered to indicate a statistically significant difference in data.

To study the effect of SIRT3 expression on the growth of small-cell lung cancer cells, NCI-H446 cells were transfected with either a control plasmid (NC group) or a plasmid encoding SIRT3 (SIRT3 group). Western blotting and RT-PCR analysis confirmed an increase in expression of SIRT3 in cells of the SIRT3 group, when compared with those of the NC group (P<0.01), indicating that the transfection was successful. A microscopy-based analysis of cell morphology revealed an increase in cell death among cells of the SIRT3-group at 24 h post-transfection (Fig. 1B). By 48 h post-transfection the difference in cell death between the control and SIRT3 groups was even more pronounced. SIRT3 overexpression promoted cell rounding and detachment, resulting in increased numbers of cells floating in the culture media. MTT assays revealed that cell viability in the SIRT3 group was reduced to 75% compared to the control group 24 h after transfection (Fig. 1C). By 48 and 72 h post-transfection, viability of the SIRT3 group was reduced to 43 and 20%, respectively, indicating that overexpression of SIRT3 in lung cancer NCI-H446 cells inhibited the growth of NCI-H446 cells in lung cancer.

Cell apoptosis was detected by flow cytometry BD Accuri C6. The upper right quadrant represents late apoptosis and necroptosis, and the lower right quadrant represents early apoptosis. It can be observed from Fig. 2A that early apoptosis of the SIRT3 group was increased compared to the NC group, and late apoptosis and necroptosis were also increased (P<0.01). The mitochondrial membrane potential was also examined by flow cytometry, and it was revealed to be significantly decreased in the SIRT3-overexpressing cells when compared with the control (Fig. 2B; P<0.05). The results of western blotting experiments revealed that the expression of the apoptosis-related proteins cleaved caspase-9, Bax, Bid, and cleaved caspase-3 was significantly increased when compared with controls (Fig. 2C; P<0.01), and that the expression of the anti-apoptotic proteins Bcl-2 and MCL1 was significantly decreased (Fig. 2C; P<0.01). RT-PCR analysis also revealed a significant increase in the expression of the pro-apoptotic genes TP53, BAX and CASP3 (P<0.05) and a decrease in the expression of the pro-survival gene BCL2 (P<0.01) in SIRT3-overexpressing cells (Fig. 2D). Furthermore, after 24 h of treatment with SIRT3 overexpression, a significant increase in caspase-3/7 activity was revealed in NCI-H446 cells (Fig. 2E).

Nec-1, an inhibitor of necrotic apoptosis, was used to determine whether SIRT3-dependent cell death involved the necrotic pathway. Twenty-four hours post-transfection with control or SIRT3 expression plasmids, NCI-H446 cells were treated with different concentrations of Nec-1, and cell survival was subsequently assessed by MTT assay. This experiment revealed that Nec-1 treatment could partially reverse the effects of SIRT3-overexpression on cell death (Fig. 3A; P<0.01). However, the effects of Nec-1 treatment did not increase with increasing drug concentration. Western blot analysis of the expression of RIP1, RIP3, MLKL and p-MLKL, four important regulators of programmed necroptosis, revealed that their expression was significantly increased in SIRT3-overexpressing cells, when compared with controls (Fig. 3B; P<0.01). Collectively, these data indicated that SIRT3 overexpression promoted necroptosis in NCI-H446 cells.

Next, mutant p53 expression in SIRT3-overexpressing NCI-H446 cells was evaluated by western blotting. As revealed in Fig. 4A, mutant p53 protein levels were significantly decreased in cells overexpressing SIRT3, when compared with controls (P<0.05). SIRT3 overexpression resulted in a reduction in the half-life of mutant p53 protein (Fig. 4B). Therefore, the mechanism underlying this SIRT3-dependent decrease in mutant p53 expression was investigated. Protein degradation in eukaryotic cells is primarily controlled by the lysosomal and ubiquitin proteasome pathways. To determine whether the lysosomal pathway was implicated in mutant p53 degradation, the expression of the autophagy-associated proteins LC3 II and Beclin-1 were examined in SIRT3-overexpressing cells and it was revealed that their levels remained unchanged, when compared with the control NC group (Fig. 4C). Therefore, it was hypothesized that mutant p53 was degraded by the ubiquitin proteasome pathway.

MG132 is a potent inhibitor of protein degradation mediated by the ubiquitin proteasome pathway (13). SIRT3-overexpressing NCI-H446 cells were treated with MG132 for 24 h, and then protein lysates were prepared for analysis of mutant p53 expression. Western blot analysis revealed that mutant p53 protein expression in the NC+MG132 group was significantly increased, when compared with the NC group (Fig. 4D; P<0.05). An interaction between mutant p53 and SIRT3 was demonstrated by immunoprecipitation assay (Fig. 4E). Immunoprecipitation assays also revealed that mutant p53 in SIRT3-overexpressing cells was more highly ubiquitinated than in control cells. These data indicated that overexpression of SIRT3 promoted degradation of mutant p53 by the ubiquitin proteasome pathway.

To further study the effects of SIRT3 on NCI-H446 cells, an in vivo experiment was conducted. NCI-H446 cells were injected subcutaneously into mice, and tumors were then allowed to establish and grow until approximately 5 mm in diameter. After 25 days, mice and tumors were photographed and differences in tumor size were evaluated. SIRT3 overexpression was revealed to inhibit tumor growth in vivo (Fig. 5A and B). The tumor growth index also confirmed that SIRT3 inhibited tumor growth (Fig. 5C). H&E staining revealed increased apoptosis in the tumor tissues of the SIRT3 group (Fig. 5D). Immunohistochemical analysis demonstrated that SIRT3 protein expression was enhanced and that mutant p53 expression was decreased in the SIRT3 group, when compared with the PQ control group (Fig. 5E). Western blot analysis of tumor tissue extracts confirmed that SIRT3 treatment resulted in reduced mutant p53 expression (Fig. 5F), a finding that was consistent with our in vitro data, and which indicated that SIRT3 inhibited the development of small-cell lung cancer. Analysis of the expression of apoptosis-related proteins by western blotting revealed that the ratio of Bcl-2 to Bax protein in SIRT3 tumors was reduced, when compared with PQ control tumors (Fig. 5G). Cleaved caspase-3 expression was also upregulated in SIRT3 tumors. In addition, RT-PCR was used to evaluate changes in the expression of apoptosis-related genes (Fig. 5H). In agreement with the protein expression data, a decrease in BCL2 to BAX gene expression, and increased CASP3 gene expression was observed in SIRT3 tumors, when compared with controls.