The tumor tissues and corresponding paracancerous tissues of 68 patients with PC treated between March 2016 and March 2017 in The First Hospital of Changsha (Changsha, China) were collected. All patients did not receive radiotherapy and chemotherapy before operation. Written informed consent was provided by all participants. The present study was approved [(2016) Ethical Review (Clinical Research) approval no. 59] by the Ethics Committee of The First Hospital of Changsha (Changsha, China).

SW1990 (cat. no. CRL-2172), BXPC-3 (cat. no. CRL-1687) and PANC-1 (cat. no. CRL-1469) cells were purchased from the American Type Culture Collection. PC-2 (cat. no. JK-CS1579) and human normal pancreatic epithelial (HPDE; cat. no. JK-CS2033) cells were obtained from Shanghai Jingkang Bioengineering Co., Ltd. The cells were cultured in a constant temperature incubator with 5% CO₂ at 37˚C. The medium used was DMEM containing 10% fetal bovine serum (both from Gibco; Thermo Fisher Scientific, Inc.).

PcDNA-SIRT6, vector, small interfering (si)-SIRT6#1 (5'-CCAAGUGUAAGACGCAGUATT-3'), si-SIRT6#2 (5'-TCATGACCCGGCTCATGAA-3'), si-SIRT6#3 (5'-CGAGGAUGUCGGUGAAUUA-3') and si-NC (5'-GGCCAAGCCUUGUGUAAAU-3') were obtained from Shanghai GenePharma Co., Ltd. PANC-1 or SW1990 cells were inoculated into six-well plates and cultured overnight. According to the manufacturer's protocol of Lipofectamine^™ 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.), the cells were mixed with the corresponding plasmids, incubated for 6 h in an incubator with 5% CO₂ at 37˚C, and then cultured with fresh medium for 24 h. The concentration of siRNAs was 100 nM and the mass of vector was 2 µg. After 48 h, the overexpression of SIRT6 in PANC-1 cells and downregulation of SIRT6 in SW1990 cells were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.

The transfected PANC-1 cells were inoculated into 96-well plates at a concentration of 3,000 cells/well. After 48 h of culture, 10 µl of CCK-8 solution (Beyotime Institute of Biotechnology) was added to each well and the cells were incubated for 2 h at an atmosphere containing 5% CO₂ at 37˚C. The absorbance value at the wavelength of 450 nm was detected using a microplate reader (BioTek Instruments, Inc.).

Total RNA of tissues and cells (PANC-1 and SW1990) was isolated using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), and then single stranded cDNA was obtained according to the instructions of Prime ScriptTM RTMaster Mix (Takara Biotechnology Co., Ltd.). Subsequently, the RT-qPCR experiment was carried out according to the protocol of SYBR Premix Ex TaqTM II kit (Takara Biotechnology Co., Ltd.). The expression levels of mRNA were quantified using the 2^-ΔΔCq method (32). GAPDH was used as an internal reference gene. The thermocycling conditions for qPCR were as follows: 95˚C for 5 min, and then 40 cycles (95˚C for 10 sec, 60˚C for 30 sec). The following primer pairs were used for qPCR: SIRT6 forward 5'-CAAGTGTAAGACGCAGTACG-3' and reverse, 5'-GATGGTGTCCCTCAGCTCTC-3'; and GAPDH forward, 5'-TGACTTCAACAGCGACACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'.

The PANC-1 and SW1990 cells were inoculated into six-well plates at a density of 2x10⁵/well and cultured for 24 h at 37˚C and 5% CO₂. DCFH-DA (50 µM) (Beyotime Institute of Biotechnology) was added to each well and cells were cultured for 30 min at 37˚C and 5% CO₂. Subsequently, the supernatant was discarded and the cells were washed with PBS for 3 times. Finally, the fluorescence intensity was measured under a fluorescence microscope (Nikon eclipse 80i; Nikon Corporation). The excitation and emission wavelengths were 488 and 525 nm, respectively.

The Fe²⁺ level in PANC-1 and SW1990 cells was detected by Fe²⁺ colorimetry detection kit (catalog no. E1042-100; Applygen Technologies, Inc.). The cells were lysed using RIPA reagent, and the supernatant was collected by centrifugation (10,000 x g, 4˚C, 10 min). Reagent A was obtained by mixing the buffer in the kit and 4.5% potassium permanganate solution at 1:1 ratio. Then, the supernatant was mixed with reagent A at 1:1 ratio according to the manufacturer's instructions and was incubated at 60˚C for 1 h. After cooling at room temperature, Fe²⁺ content was detected using Fe²⁺ detection agent (30 µl, 30 min) in the kit. The absorbance value at 550 nm wavelength was detected using a microplate reader (BioTek Instruments, Inc.).

Erastin (catalog no. E7781) and ferrostatin-1 (catalog no. S7243) were obtained from Selleck Chemicals. The PANC-1 cells transfected with pcDNA-SIRT6 and the SW1990 cells transfected with si-SIRT6 were treated with erastin (10 µM) or ferrostatin-1 (1 µM) for 24 h, and then the Fe²⁺ level was evaluated.

Total protein of tissues or cells (PANC-1 and SW1990) was extracted using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein content was detected using BCA detection kit (Beyotime Institute of Biotechnology). A total of 30 µg protein sample/well was separated using 10% SDS-PAGE and then transferred to PVDF membranes (MilliporeSigma). Following that, the membranes were incubated for 1 h with 5% skimmed milk powder at room temperature. The membranes were incubated overnight at 4˚C with the corresponding primary antibodies: SIRT6 (1:1,000; catalog no. ab119007), NF-κB p65 (1:1,000; catalog no. ab32536), lamin B (1:1,000; catalog no. ab232731), IκBα (1:1,000; catalog no. ab76429), GPX4 (1:1,000; catalog no. ab125066), SLC7A11 (1:1,000; catalog no. ab37185), HK2 (1:1,000; catalog no. ab104836) and lactate dehydrogenase A (LDHA; 1:1,000; catalog no. ab134187) (all from Abcam). Following the primary incubation, membranes were washed three times with PBST (0.1% Tween20) and then incubated at room temperature for 2 h with the DyLight^® 488-conjugated secondary antibodies: Goat anti-rabbit IgG H&L (1:5,000; product code ab96899) or goat anti-mouse IgG H&L (1:5,000; product code ab96879; both from Abcam). Protein bands were visualized using Immobilon ECL Ultra Western HRP (MilliporeSigma). The results were analyzed using ImageJ version 6.0 (National Institutes of Health).

PANC-1 cells (1x10⁵/ml) transfected with vector or pcDNA-SIRT6 were seeded on a coverslip pre-coated with poly-L-lysine, and cultured in DMEM with or without RANKL (50 ng/ml; Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h of culture, the cells fixed with 4% cold formaldehyde at 4˚C overnight. After washing three times with PBS containing 0.1% Triton X-100, the cells were blocked with 10% bovine serum albumin (MilliporeSigma) for 2 h at room temperature, and then incubated with primary antibodies against NF-κB p65 (1:1,000; catalog no. ab288751) and DAPI (1:2,000; catalog no. ab104139) (both from Abcam) at 4˚C overnight. Cells were then incubated with Alexa Fluor 488 donkey anti-mouse immunoglobulin G (1:200; catalog no. ab150105; Abcam) secondary antibodies for 1 h at room temperature and visualized using a confocal laser-scanning microscope.

The content of glucose (catalog no. A154-1-1), lactic acid (catalog no. A019-1-1) and ATP (catalog no. A095-1-1) in energy metabolism were detected by corresponding kits (Nanjing Jiancheng Bioengineering Institute). PANC-1 cells in logarithmic growth stage were digested with trypsin, and the cell concentration was adjusted to 2x10⁵/ml with serum-free DMEM medium; cells were then inoculated on 12-well plates and kept at 1 ml per well. After 48 h of culture, the supernatant of cell culture medium was collected, and the glucose and lactic acid levels in the supernatant were determined using the glucose and lactic acid detection kits according to the manufacturer's protocol. In addition, PANC-1 cells were collected and homogenized in a hot water bath (90-100˚C), and then kept for 10 min in a boiling water bath. Following that, the protein concentration and ATP level were determined. In order to avoid the difference in the number of cells between the experimental groups, the content of glucose, lactic acid and ATP were examined for sample standardization by dividing the protein concentration of each sample. Glucose consumption equals to the glucose content of culture medium minus the glucose content of cell culture medium.

Animals were fed under specific-pathogen-free conditions, in the mouse-feeding facility with a 12-h light/dark cycle at 22±2˚C and 50-60% humidity, with free foraging and activity and free access to food and water. A total of 20 SPF BALB/C nude mice were randomly divided into two groups [SIRT6 group (n=10) and vector group (n=10)]. PANC-1 cells (1x10⁶) in 200 µl phosphate-buffered saline were subcutaneously inoculated in nude mice of each group. The tumor volume was recorded every 7 days. After 4 weeks, the nude mice were euthanized by intraperitoneal injection of 120 mg/kg pentobarbital sodium, and the tumor was stripped and weighed. During the experiment, when dyspnea, diarrhea, incontinence, rapid weight loss, or loss of appetite (more than 24 h without eating and drinking) were observed, the mice should be euthanized. The death of mice was determined by observing the cardiac arrest and pupil dilation. Animal health and behaviour were monitored every 3 days. The animal study was approved [(2016) Ethical Review (Clinical Research) approval no. 59] by the Institutional Animal Care and Use Committee of The First Hospital of Changsha (Changsha, China).

The tumor tissue was cut into 4.5-µm-thick sections after dehydration, transparency and paraffin embedding. The sections were dewaxed at 60˚C for 20 min with xylene and hydrated for 5 min with absolute, 95 and 80% ethanol solutions, respectively. The slices were soaked for 20 min using 3% hydrogen peroxide, and then placed in a citrate buffer at 95˚C and cooled to room temperature. The sections were blocked for 45 min with 5% bovine serum albumin (MilliporeSigma) at 37˚C. Subsequently, tissue sections were incubated overnight at 4˚C with the following primary antibodies: Ki67 (1:200; product code ab15580), SIRT6 (1:200; product code ab236024), GPX4 (1:100; product code ab125066), SLC7A11 (1:200; product code ab37185), HK2 (1:200; product code ab104836) and LDHA (1:100; product code ab134187; all from Abcam). Following the primary incubation, the sections were incubated for 30 min at 37˚C with the following secondary antibodies: goat anti-rabbit IgG H&L (1:250) or goat anti-mouse IgG H&L (1:250). After DAB staining for 3-5 min at room temperature, the nuclei were counterstained with hematoxylin for 3 min at room temperature. The sections were differentiated with 1% hydrochloric-alcohol, returned to blue with 1% ammonia, dehydrated and made transparent, dried in the air and sealed with neutral resin. Images were captured under a light Olympus BX-41 microscope (Olympus Corporation).

SPSS 30.0 software (IBM Corp.) was used to analyze the data, and the data was expressed as the means ± SD. Paired Student's t-test was used for the comparison between the tumor and corresponding paracancerous tissues, and the remaining two group comparisons were analyzed by unpaired Student's t-test. One-way ANOVA followed by Bonferroni test was used for the comparison among multiple groups. The χ² test was used to analyze the relationship between SIRT6 expression and clinicopathological characteristics. Kaplan-Meier analysis followed by the log-rank test (https://kmplot.com/analysis/) was used to estimate the overall survival of PC patients. P<0.05 was considered to indicate a statistically significant difference.

The expression of SIRT6 in PC tissues and cells was detected. The results of RT-qPCR and western blotting revealed that the expression of SIRT6 in PC tissues was lower than that in corresponding adjacent tissues (Fig. 1A and D). Moreover, SIRT6 was significantly downregulated in PC cell lines (SW1990, PC-2, BxPC-3 and PANC-1) compared with HPDE cells (Fig. 1E). In the present experiments, in order to study the effect of upregulation and downregulation of SIRT6 on PC cells, SIRT6 was overexpressed in the low-expressing line (PANC-1 cells), and SIRT6 was silenced in the high-expressing line (SW1990 cells). In addition, our clinical and database data indicated that patients with PC with low expression of SIRT6 had a lower survival rate than the patients with high expression of SIRT6 (Fig. 1B). Furthermore, Kaplan-Meier survival curve analysis showed that patients with PC with low expression of SIRT6 had poor survival, whereas high expression of SIRT6 was positively associated with overall survival (HR=0.54, 95% CI: 0.35-0.84, P=0.0052, Fig. 1C). In addition, our clinical data indicated that the expression of SIRT6 was negatively associated with the distant metastasis, histological grade and TNM stages in patients with PC (Table I). These results suggested that the expression of SIRT6 may affect the progression of PC.

In order to explore the function of SIRT6 in PC cells, SIRT6 was upregulated by transfection of pcDNA-SIRT6 into PANC-1 cells. The results of RT-qPCR and western blot analysis verified that SIRT6 was overexpressed in PANC-1 cells transfected with pcDNA-SIRT6 (Fig. 2A and B). The results of CCK-8 showed that the overexpression of SIRT6 inhibited the viability of PANC-1 cells at 48 and 72 h (Fig. 2C). The enhancement of SIRT6 could increase the level of Fe²⁺ in PANC-1 cells (Fig. 2D). The addition of erastin (ferroptosis inducer) further increased the level of Fe²⁺, but the addition of ferrostatin-1 (ferroptosis inhibitor) reversed that effect (Fig. 2E). Moreover, SIRT6 elevation increased the level of ROS (Fig. 2F) and weakened the expression of GPX4 and SLC7A11 in PANC-1 cells (Fig. 2G). In addition, overexpression of SIRT4 inhibited the glycolysis through inhibiting the production of lactic acid and ATP and reducing glucose uptake in PANC-1 cells (Fig. 2H-J). The expression of HK2 and LDHA was also inhibited (Fig. 2K). These results demonstrated that SIRT6 upregulation increased ferroptosis and inhibited glycolysis in PC cells.

SIRT6 was downregulated by transfection of si-SIRT6 (#1, #2 and #3) in SW1990 cells, which was used to further verify the role of SIRT6 in PC cells. The silencing of SIRT6 in SW1990 cells transfected with si-SIRT6 was confirmed by RT-qPCR and western blotting (Fig. 3A and B). The relative expression of SIRT6 was the lowest in si-SIRT6#1-transfected SW1990 cells, which was selected for the subsequent experiments. The silencing of SIRT6 enhanced the viability of SW1990 cells at 48 and 72 h (Fig. 3C). The downregulation of SIRT6 suppressed the content of Fe²⁺ (Fig. 3D), which was enhanced by erastin and weakened by ferrostatin-1 (Fig. 3E). Furthermore, SIRT6 knockout reduced ROS levels (Fig. 3F) and increased the expression of GPX4 and SLC7A11 (Fig. 3G). In addition, the inhibition of SIRT6 promoted glucose uptake, production of lactic acid and ATP (Fig. 3H-J), and enhanced the expression of HK2 and LDHA (Fig. 3K). The aforementioned findings showed that downregulation of SIRT6 restrained ferroptosis and promoted glycolysis in PC cells.

In order to study the mechanism of SIRT6 affecting PC, the effect of SIRT6 on the transcriptional regulation of NF-κB p65 was explored. The results of western blot analysis showed that overexpression of SIRT6 increased the expression of IκBα in the cytoplasm, whereas it inhibited the expression of p65 in the nucleus (Fig. 4A). In addition, the results of immunofluorescence assay about p65 expression in the nucleus were consistent with those of western blotting (Fig. 4B). Receptor activator of nuclear factor κΒ ligand (RANKL), as an agonist of NF-κB p65 nuclear transfer, is commonly used in the study of the NF-κB signaling pathway. The results indicated that RANKL reversed the effects of SIRT6 upregulation on the level of IκBa (cytoplasm) and p65 (nucleus) (Fig. 4C). Cell viability experiments revealed that the decrease of the viability of PANC-1 cells induced by SIRT6 was weakened by RANKL (Fig. 4D). In addition, RANKL blocked the upregulation of ROS in PANC-1 cells induced by SIRT6 (Fig. 4E). Overexpression of SIRT6 inhibited the expression of GPX4, SLC7A11, HK2 and LDHA, whereas RANKL reversed these effects (Fig. 4F). These results indicated that SIRT6 inhibited tumor characteristics of PC by activating the NF-κB signaling pathway.

To confirm the oncological properties of SIRT6 in vivo, a xenografted tumor model was established by injecting PANC-1 cells subcutaneously into nude mice. The results showed that the upregulation of SIRT6 could inhibit the volume of tumor (Fig. 5A) and had no effect on the body weight of mice (Fig. 5B). Concurrently, the weight of the tumor was also reduced by SIRT6 overexpression (Fig. 5C). Immunohistochemical results revealed that SIRT6 was upregulated, and the expression levels of Ki67, GPX4, SLC7A11, HK2 and LDHA were inhibited in the xenografted tumor tissues of mice of the SIRT6-overexpression group (Fig. 5D). Moreover, the upregulation of SIRT6 increased the cytoplasmic level of IκBa and suppressed the nuclear level of p65 (Fig. 5E). These results suggested that SIRT6 may be an antineoplastic gene in PC by promoting ferroptosis and reducing glycolysis.