Human lung cancer A549 cells (cat. no. CCL-185™; American Type Culture Collection) were cultured at 37°C in a humidified atmosphere with 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.). The cell line was authenticated using short tandem repeat profiling and mycoplasmas were tested to confirm that the cells were free of contamination.

TP (≥98%) and actinomycin D (Act D, 20 ng/ml) were obtained from Sigma-Aldrich (Merck KGaA). These drugs were aliquoted upon delivery, stored at 100 µg/ml in DMSO at −80°C, and diluted to the indicated concentrations using serum-free culture medium when used. Cells were treated with Act D for 36 h prior to relevant experiments.

Cells were treated with TP at 12.5, 50 and 100 ng/ml for 36 h. Apoptosis, cell cycle and cell viability were detected according to the manufacturer's instructions. For apoptosis, 5×10⁵ cells were collected with EDTA-free trypsin, washed with cold PBS, incubated with 5 µl Annexin V-FITC and 10 µl propidium iodide (PI; 20 µg/ml) at room temperature for 15 min in the dark, and analyzed by a FACS Canto II instrument (BD Biosciences); the apoptotic rate was calculated as the sum of early + late apoptotic cells.

For cell cycle analysis, 5×10⁵ cells were fixed with absolute ethanol at −20°C overnight, washed with cold PBS, incubated with 5 mg/ml RNase at 37°C for 30 min, stained with 5 mg/ml PI in the dark at room temperature for 30 min, and last analyzed by a FACS Canto II. Flow cytometry data were analyzed with FlowJo 7.6 software (FlowJo LLC).

For cell viability, 3×10³ cells were seeded in 96-well plates and cultured with 100 µl DMEM. Cell Counting Kit-8 solution (Dojindo Molecular Technologies, Inc.) was incubated with cells at 37°C for 3 h. Absorbance at 450 nm was measured, and cell viability was evaluated according to the manufacturer's instructions.

TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total cellular RNA. RT was conducted using a PrimeScript RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions. Amplification of 45S and 18S rRNA was performed using a C1000 Thermal Cycler detection system (Bio-Rad Laboratories, Inc.) in triplicate using 1 ng of cDNA, 125 nM forward and reverse primer and 25 µl 2X SYBR^® Premix Ex Taq™ (Takara Bio, Inc.) in a 50-µl reaction. The reaction parameters were 95°C for 1 min, followed by 42 cycles of 95°C for 15 sec, 56°C for 25 sec and 72°C for 30 sec. The following primers were used: 45S forward, 5′-GCGGAACCCTCGCTTCTC-3′ and reverse, 5′-CTCCGTTATGGTAGCGCTGC-3′; 18S forward, 5′-CTCTCCGGAATCGAACCCTGA-3′ and reverse, 5′-CGACGACCCATTCGAACGTCT-3′; and GAPDH forward, 5′-AGCCACATCGCTCAGAACAC-3′ and reverse, 5′-GAGGCATTGCTGATGATCTTG-3′. The relative mRNA expression levels were quantified using the 2^−∆∆Cq method (18); GAPDH was used as the internal reference.

Cells were washed and scraped with cold PBS, centrifuged at 16,000 × g at 4°C for 15 min, and suspended in IP lysis buffer (Cell Signaling Technology, Inc.). Protein concentrations were measured using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.). Protein (100 µg) was incubated with anti-MDM2 (1:100; cat. no. 86934; Cell Signaling Technology, Inc.) or IgG (1:500; cat. no. sc-2025; Santa Cruz Biotechnology, Inc.) antibodies mixed with 500 µl of TBS-0.5% Tween 20 (TBST) along with 10 µl of Dynabeads protein G on a rotating platform at 4°C overnight. Protein beads were then washed three times with 0.6 ml of ice-cold TBST buffer. A micropipette was used to remove the last traces of buffer. Antibody-protein complexes were eluted with 1X SDS sample buffer, electrophoresed and subjected to western blotting.

TP-treated cells were collected with Cell Lysis Buffer (cat. no. 9803S; Cell Signaling Technology, Inc.) supplemented with phenylmethanesulfonylfluoride fluoride (cat. no. 8553S; Cell Signaling Technology, Inc.) at the indicated times. The BCA assay kit was used to determine the protein concentrations. Proteins (30 µg) were denatured in boiling water for 10 min and fractioned by using SDS-PAGE with 10% denaturing acrylamide gels. The fractioned proteins were transferred to polyvinylidene fluoride membranes. The blotted membranes were blocked with 5% nonfat milk in TBST for 1 h at room temperature, followed by incubation with the diluted primary antibodies against MDM2 (1:1,000; cat. no. 86934, Cell Signaling Technology, Inc), phosphorylated (p)-p53 (1:1,000; cat. no. 2521; Cell Signaling Technology, Inc), cleaved (c)-caspase 9 (1:1,000; cat. no. 20750; Cell Signaling Technology, Inc), c-caspase 3 (1:1,000; cat. no. 9664; Cell Signaling Technology, Inc), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), RPL23 (1:3,000, cat. no. ab241088; Abcam), p53 (1:5,000; cat. no. 10442-1-AP; ProteinTech Group, Inc.), p53 upregulated modulator of apoptosis (1:1,000; PUMA; cat. no. 55120-1-AP; ProteinTech Group, Inc.) and BCL2 (1:1,000; cat. no. 12789-1-AP; ProteinTech Group, Inc.) at 4°C for overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. nos. 7076 and 7074; Cell Signaling Technology, Inc.) for 1.5 h at room temperature. Enhanced chemiluminescence reagents (EMD Millipore) were used to visualize the immunoreactive proteins, and densitometric analysis was performed by using ImageJ v1.8.0 software (National Institutes Of Health).

Cells were seeded into confocal microscopy dishes, treated with 50 ng/ml TP for 24 h and fixed in 4% paraformaldehyde at room temperature for 20 min. Next, 0.2% Triton X-100 was used to permeabilize the membranes. The prepared cells were washed with cold PBS and blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) at room temperature for 30 min. Primary antibodies against nucleophosmin (B23; 1:200; cat. no. ab15440; Abcam; or 1:100; cat. no. 10306-1-AP; ProteinTech Group, Inc.), RNA Pol I (1:100; cat. no. sc-48385; Santa Cruz Biotechnology, Inc.), UBF (1:100; cat. no. sc-13125; Santa Cruz Biotechnology, Inc.) and nucleolin (NCL; 1:1,000; cat. no. 14574; Cell Signaling Technology, Inc.) were added and incubated at 4°C overnight. Dylight™ 488 (green) or 594 (red)-conjugated secondary antibodies (1:500; cat. nos. A23210 and A23420; Abbkine Scientific Co., Ltd.) were applied for 1 h at room temperature. DAPI (Thermo Fisher Scientific, Inc.) was used to counterstain the nuclei. Immunofluorescent images of the prepared cells were viewed under a fluorescence laser-scanning confocal microscope (magnification, ×63; Carl Zeiss AG) in 20 randomly selected fields.

Cells were seeded on coverslips and incubated with DMEM for adherence. Then, 50 ng/ml TP was used to stimulate the cells for 24 h. Next, 2 mM 5-FU (Sigma-Aldrich; Merck KGaA) was incubated with the cells growing on coverslips at room temperature for 15 min. The processes of washing, fixation and membrane permeabilization were performed as described in the Immunofluorescence assay section. Specific monoclonal antibodies recognizing halogenated uridine (1:400; cat. no. B8434; Merck KGaA) and NCL (cat. no. 14574; Cell Signaling Technology, Inc.) were used to incubate with the prepared cells at 4°C overnight. Dylight-conjugated secondary antibody incubation, nuclear counterstaining and immunofluorescent imaging were conducted as previously described.

ChIP assays were performed according to the manufacturer's instructions (EZ ChIP™ kit; cat. no. 17-371; EMD Millipore). Briefly, the prepared cells were cross-linked with 1% formaldehyde for 10 min at room temperature. The unreacted formaldehyde was quenched using 0.125 M glycine. The cells were washed with cooled PBS containing protease inhibitor cocktail II included in the kit for 5 min, and then scraped and pelleted. The cell pellets were lysed in 1% SDS lysis buffer. Sonication (pulse for 5 sec, pause for 9 sec, cycle 23 times) on wet ice (0°C) was conducted for DNA fragmentation. An aliquot of the sheared lysate was preserved at 4°C until use as an input control. Sheared lysate (100 µl) was diluted with dilution buffer and incubated with anti-UBF (1:50; cat. no. sc-13125; Santa Cruz Biotechnology, Inc.) and anti-RNA Pol I antibodies (1:50; cat. no. sc-48385; Santa Cruz Biotechnology, Inc.) on a rotating rack at 4°C for overnight. The immunocomplexes containing DNA, protein and antibodies were captured using protein A-agarose. Immunocomplexes conjugated with beads were eluted using elution buffer including 1% SDS and 100 mM NaHCO₃ and reverse cross-linked at 65°C overnight. DNA fragments were collected, and UBF and RNA Pol I enrichment in the rDNA promoter were amplified using RT-qPCR as previously described. The primers for augmenting the rRNA promoter DNA were as follows: Forward, 5′-CGATGGTGGCGTTTTTGG-3′ and reverse, 5′-CCGACTCGGAGCGAAAGATA-3′. The negative control comprised the same assay conducted using a human normal IgG antibody (cat. no. sc-2025; Santa Cruz Biotechnology, Inc.

A549 cells (5×10⁶) were injected subcutaneously into 10 4-week-old male BALB/cAnNCr-nu/nu nude mice (18–22 g; Sippr-BK Laboratory Animal Co., Ltd.). All mice were fed with standard mouse irradiated food and tap water ad libitum, and maintained under conditions of 25°C and 50% humidity with a 12-h light/dark cycle. After 3 weeks, the mice were randomly divided into two groups of five, receiving subcutaneous TP (1.5 mg/kg) or saline (mock) injections every day for 5 weeks. The body weight and tumor volume were measured every 2 days. The tumor volume was calculated as (length × width × width)/2. Then, all mice were euthanized using carbon dioxide in according with the Guidelines for Euthanasia of Rodents Using Carbon Dioxide issued by the National Institutes of Health. After euthanasia, death was further verified by cardiac arrest and cadaveric rigidity. The animal experiments were approved by the Animal Experimentation Ethics Committee of the Tongde Hospital of Zhejiang Province.

TUNEL assays were performed to investigate the apoptotic cells in tumor tissues using an in situ cell death detection kit obtained from Roche Diagnostics. Tumor tissue was fixed with a mixture of 70% alcohol, formaldehyde and glacial acetic acid (16:1:1) at 4°C for 24 h, and paraffin-embedded tumor sections (5-µm) were dewaxed and rehydrated. Proteinase K was applied at 37°C for 25 min. Membranes were dissolved at room temperature for 20 min. Then, TdT and dUTP were mixed at 1:9 and the mixture incubated with tissues at 37°C for 2 h. DAPI solution was used to stain nuclei in the dark at room temperature for 10 min. A fluorescence microscope was used to observe and photograph the apoptotic cells. Positive cells were marked by green fluorescence. TUNEL-positive cells and total cells in sections were measured using ImageJ v1.8.0 software.

The tissue samples (5 µm) were deparaffinized and rehydrated. A timer-controlled water bath was used to conduct heat-induced antigen retrieval at 98°C for 20 min. TBS plus 0.025% Triton X-100 was used to wash the slides with gentle agitation. The slides were then blocked with 10% BSA in TBS at room temperature for 2 h. Primary antibodies against p53 (1:150; cat. no. 2527; Cell Signaling Technology, Inc.), RPL23 (1:50; cat. no. 16086-1-AP; ProteinTech Group, Inc.) and NCL (1:400; cat. no. 14574; Cell Signaling Technology, Inc.) were incubated with the samples overnight at 4°C, and 50 µl horseradish peroxidase-conjugated secondary antibody (cat. no. 8114S; Cell Signaling Technology, Inc.) was used to recognize the primary antibody at room temperature for 1 h. The tissues were then stained with 3,3′-diaminobenzidine and subsequently hematoxylin at room temperature for 4 min, and the samples were observed under a light microscope (magnification, ×40) in 20 fields of vision and quantified by ImageJ v1.8.0 software.

Experiments were performed in triplicate at least three times, independently. Data are presented as the mean ± standard deviation. One-way, two-way or mixed ANOVA followed by Tukey's, Dunnett's or Sidak's multiple comparisons test, or Student's t-test in GraphPad Prism 6 (GraphPad Software, Inc.) were used to analyze the statistical significance between groups. P<0.05 was considered to indicate a statistically significant difference.

The effects of TP were first determined on A549 cells. An Annexin V/PI staining-based assay with fluorescence-activated cell sorting was performed to analyze the population of apoptotic cells. The results showed that TP increased the apoptotic index in a dose-dependent manner (Fig. 1A). The percentage of apoptotic cells gradually increased with increasing TP concentration, from 4.1±1.34% in control cells to 12.3±1.72, 26.2±2.16 and 45.8±3.28% in TP-treated cells (Fig. 1C). Then, PI staining was performed to analyze the cell cycle (Fig. 1B). TP caused the G0/G1 fraction to decrease from 48.6±3.56% in control cells to 14.9±1.22% in 100 ng/ml TP-treated cells, and the G2/M fraction increased from 14.2±2.42% in the control to 19.2±2.68% (12.5 ng/ml TP), 25.2±3.11% (50 ng/ml TP) and 38.9±3.62% (100 ng/ml TP; Fig. 1D). These results indicated that TP arrested A549 cells at the G2/M phase. Then, the effect of TP on cell viability was evaluated. As presented in Fig. 1E, TP induced a marked dose-dependent reduction in cell viability; increasing the duration of treatment from 24 h to 48 h significantly enhanced this inhibition.

The expression and distribution of Pol I in the nucleus was investigated. Cells were treated with a negative control, TP, or a positive control, Act D, for 36 h. Act D is a well-characterized nucleolar disruptor (19). The prepared cells were dually stained with antibodies against the RNA Pol I antigen and B23; B23 is associated with ribosome biogenesis and nucleolar ribonucleoproteins (20). As presented in Fig. 2A, endogenous RNA Pol I was present primarily in the nucleolus in the control cells. Treatment with TP and Act D resulted in abnormal nuclear morphology and nucleolar disintegration, and faint traces of RNA Pol I were observed in TP-treated cells. For UBF, robust protein expression was observed in almost all nucleoli in the negative control cells; TP and Act D induced nucleolar disintegration and further diffused the distribution of UBF throughout the nucleus rather than being focused in the nucleolus (Fig. 2B). The RNA Pol I and UBF relative fluorescent intensities were significantly reduced in the TP-treated cells.

Burger et al (21) demonstrated that the disintegration of nucleolar structures is related to rRNA synthesis. Therefore, immunofluorescence assays were performed to detect whether TP plays a role in the process of RNA synthesis. NCL, a multifunctional protein that mainly regulates ribosome biogenesis and transcription, was used as a control to evaluate the integrity of the nucleoli. The results in Fig. 3A revealed that TP treatment significantly reduced the incorporation of 5-FU into nascent rRNA, suggesting that TP treatment inhibited rRNA synthesis. The ChIP assay demonstrated that TP treatment caused significant dissociation of Pol I and UBF from the promoter region of the rDNA in a dose-dependent manner (Fig. 3B and C); these results were consistent with the decreased localization of RNA Pol I and UBF observed in Fig. 2A and B. 45S rRNA is the transcriptional product driven by Pol I and UBF, and maturation of the primary transcript leads to the generation of 18S rRNA (16). RT-qPCR analysis was performed to determine the effect of TP on primary rRNA transcript 45S and modified 18S. The results in Fig. 3D showed that TP significantly reduced the expression of 45S pre-rRNA in a dose-dependent manner, and the expression of 18S expression was also significantly impaired. This result indicated that TP inhibited rRNA synthesis by inhibiting Pol I and UBF, thus further interfering with rRNA processing.

When ribosome biogenesis is interrupted, RPs may not be used for ribosome biogenesis and can bind to MDM2, which inhibits MDM2 from mediating p53 degradation (22,23). To determine whether there are intrinsic links between TP-induced ribosome biogenesis disorder and cell survival inhibition, A549 cells were treated with TP or a positive control, and co-IP assays were carried out with an anti-MDM2 antibody to pull down the associated proteins. The results showed that TP treatment increased the binding of RPL23 to MDM2 and reduced the binding of p53 to MDM2 (Fig. 4A). These results suggested that there may be competitive binding of RPL23 and p53 to MDM2. The increased binding of RPL23 to MDM2 led to reduced binding of p53, resulting in decreased MDM2-mediated degradation of p53.

p53 stabilization can evoke signaling events involved in cell survival, such as apoptotic and cell cycle pathways (24). As shown in Fig. 4B and C, TP treatment induced increases in p-p53, and activation of caspase 9 and caspase 3. Reduced levels of BCL2 were also induced by TP and the positive control. These results suggested that TP induced apoptosis and cell cycle arrest via p53 activation.

To further validate the effects of TP on A549 cell survival, A549 ×enografts were implanted in nude mice. BALB/c mice were injected subcutaneously on their right limb. At 3 weeks later, the mice were divided randomly into two groups of five mice. Each group was treated with saline or TP once a day for 4 weeks. The results showed that TP treatment notably inhibited tumor growth from day 13 (Fig. 5A and B). Moreover, the body weights of the TP-treated mice were significantly higher than that of the control group (Fig. 5C). TP treatment induced enhanced expression of dUTP, which binds mainly to the terminal 3-OH of fragmented DNA, indicating that apoptosis in TP-treated xenografts was markedly activated (Fig. 5D and E). Consistent with the in vitro experiments, immunohistochemistry revealed robust p53 expression in response to TP administration (Fig. 6A and B). In the control xenografts, NCL was found predominantly in the nucleolus; however, TP treatment caused it to disperse to the nucleus (Fig. 6A), suggesting that the nucleolus was disrupted. NCL and RPL23 exhibited nearly the same expression intensity between the two groups (Fig. 6C and D); however, TP treatment resulted in RPL23 being more likely to be located at the edge of the cell membrane (Fig. 6A).