The TC-1 cell line was generated by in vitro co-transfection of murine lung C57BL/6 cells with HPV16 E6/E7 and activated human H-Ras (G12V) oncogenes (25). In the present study, TC-1 cells were maintained in RPMI 1640 medium (MilliporeSigma) supplemented with 10% foetal calf serum (FCS; Gibco; Thermo Fisher Scientific, Inc.), 2 mM L-glutamine (MilliporeSigma) and antibiotics (100 U/ml gentamicin and 40 µg/ml nystatin; MilliporeSigma) under standard conditions (5% CO₂, 37°C, 95% relative humidity). The TRAMP-C2 murine prostate cancer cell line was derived from a heterogeneous primary tumour in the prostate of PB-Tag C57BL/6 mice (26). In the present study, TRAMP-C2 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; MilliporeSigma) supplemented with 5% FCS, 5% Nu-Serum IV (BD Biosciences), 5 µg/ml human insulin (MilliporeSigma), dehydroisoandrosterone (10 nM; MilliporeSigma), 100 U/ml gentamicin and 40 µg/ml nystatin in standard conditions (5% CO₂, 37°C, 95% relative humidity). The human breast adenocarcinoma MDA-MB-231 cell line was cultured in high-glucose (4.5 g/l) DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin sulphate (Gibco; Thermo Fisher Scientific, Inc.) under standard conditions (5% CO₂, 37°C, 95% relative humidity). Mycoplasma testing was performed with a negative result for all cell lines. All cell lines were purchased from the American Type Culture Collection.

Stattic was purchased from MedChemExpress. Stattic analogues, K1823 (24) and K1836, were synthesized by the Department of Chemistry, Faculty of Sciences, University of Hradec Kralove (Hradec Kralove, Czech Republic). The compounds' chemical formulae are presented in Fig. 1. Chemical synthesis started from benzo[b]thiophene, which was oxidized to corresponding benzo[b]thiophene-1,1-dioxide using 3-chloroperoxybenzoic acid as an oxidative agent in dichloromethane. In the next step, C6 was modified by reaction with nitrating mixture HNO₃/H₂SO₄, which produced 6-nitrobenzo[b]thiophene-1,1-dioxide. The nitro group was reduced using Fe/NH₄Cl as a specific reductant in neutral medium (MeOH/H₂O) to synthesize the necessary amine building block. Compound K1823 was synthesized using a palladium-catalysed Buchwald-Hartwig C-N cross-coupling reaction of 6-amino benzo[b]thiophene 1,1-dioxide and 1-bromo-3-(trifluoromethyl)benzene that were stirred in toluene with Cs₂CO₃ and catalytic amounts of racemic-2,2′-bis(diphenylphosphino)-1,1′-binaphthalene and Pd(OAc)₂ for 3 days at 120°C, which yielded the final amine (K1823; 33% yield).

The reactive acyl chloride was prepared overnight from 1H-pyrrolo[2,3-b]pyridine-4-carboxylic acid using (COCl)₂ in dichloromethane with a catalytic amount of dimethylformamide (DMF). Prepared 1H-pyrrolo[2,3-b]pyridine-4-carboxylic acid chloride was treated with 6-aminobenzo[b]thiophene 1,1-dioxide in triethylamine (as a base), which generated NH-nucleophiles and produced the final amide (K1836) at 10% yield after two steps. Based on high pressure liquid chromatography analysis with UV detection (λ, 254 nm), the non-calibrated purity of the K1823 and K1836 final products was >95%. Nuclear magnetic resonance spectra of the K1823 and K1836 compounds are presented in Fig. S1, Fig. S2, Fig. S3, Fig. S4.

The antibodies were used for western blotting were as follows: Rabbit monoclonal pSTAT3 (Y705; 1:2,000; cat. no. 9145S; Cell Signaling Technology, Inc.), mouse monoclonal STAT3 (1:1,000; cat. no. 9139S; Cell Signaling Technology, Inc.), rabbit monoclonal GAPDH (1:10,000; cat. no. 2118S; Cell Signaling Technology, Inc.). The secondary antibodies used were as follows: Goat IgG-HRP anti-rabbit (1:2,000; cat. no. 7074S; Cell Signaling Technology, Inc.) and goat IgG-HRP anti-mouse (1:10,000; cat. no. ab6789; Abcam).

To induce senescence in tumour cells, either TC-1 or TRAMP-C2 cells were cultured in fresh medium for 24 h, 7.5 µM DTX (Actavis LLC) was then added to the culture. The dose of DTX was selected as the dose able to induce senescence but not apoptosis based on previous studies (7,22). Tumour cells were cultured in the medium containing DTX for 4 days. Senescence in tumour cells was confirmed by assessment of senescence-associated β-galactosidase (SA-β-gal) activity using a SA-β-gal Staining Kit (Cell Signaling Technology, Inc.) according to the manufacturer's instructions. Images of cell cultures were captured using an inverted Leica DMI 8 light microscope (Leica Microsystems GmbH). In the experiments comparing proliferating and senescent cells, after 4-day induction of senescence, cells were washed twice with PBS to remove DTX in the medium to avoid its possible influence on the results. K1836 was thereafter added for 72 h with repeated supplementation every 24 h as post-DTX. In some experiments, K1836 was added 6 h before DTX as pre-DTX. Detailed analyses describing the phenotype of the DTX-induced senescent murine and human cells have been previously published (7,22,23).

The proportion of live, apoptotic or necrotic cells were assessed using propidium iodide (PI) and Annexin V staining. Cells were harvested and centrifuged (4°C, 10 min, 180 × g); the pellet was washed in cold PBS, centrifuged (4°C, 10 min, 180 × g) and resuspended in Annexin V Binding Buffer (ApoFlowEx FITC Kit; cat. no. D7044, EXBIO Praha, a.s.). Annexin V-FITC and PI were added, mixed and incubated at room temperature in the dark for 15 min. After incubation, the cells were centrifuged (4°C, 10 min, 180 × g), resuspended in Annexin V Binding Buffer and then analysed using a BD FACS Symphony™ flow cytometer (BD Biosciences). Data were analysed using FlowJo 10 software (FlowJo LLC).

Cells were seeded at a density of 5,000 cells/well in 96-well F microplates (Nunc; Thermo Fisher Scientific, Inc.). After 24 h of incubation at 37°C, the test compounds Stattic, K1823 and K1836 (dissolved in DMSO as 50 mM stock) were added to the cells in increasing concentrations 0–50 µM. MTT and DMF were added 24 and 30 h later, respectively. The absorbance was measured at 560 nm after 24 h of incubation at 37°C following DMF addition using a Thermo Scientific Multiskan EX (Thermo Fisher Scientific, Inc.).

Cells were seeded in white, cell culture-treated, solid 384-well plates (Corning Inc.) at 1,000 cells/well in 20 µl media. Test compounds Stattic, K1823 and K1836 were diluted in DMSO and added to the cells using contact-free acoustic transfer using an Echo^® 655 Liquid Handler (Labcyte, Inc.; Beckman Coulter, Inc.) integrated in a fully automated robotic ACell HTS station (HighRes Biosolutions). Compounds were tested in the range 0.045–100 µM, in triplicate. Cells were incubated with the compounds for 72 h at 37°C. Cell viability was evaluated by assessing the level of intracellular ATP using a CellTiter-Glo^® (Promega Corporation) luminescent assay according to the manufacturer's protocol. The luminescent signal was quantified in an EnVision multimode plate reader (PerkinElmer, Inc.). Data were collected, normalized and processed using the ScreenX LIMS system (in-house developed proprietary system; http://www.openscreen.cz/en/screenx).

Proliferating cells were seeded and incubated for 24 h, Stattic, K1823 and K1836 were added for 4 h and then cells were washed with cold PBS, harvested and lysed with 95°C heated modified Laemmli sample lysis buffer (1% SDS, 60 mM Tris-HCl, 10% glycerol and 0.4% β-mercaptoethanol in distilled water). Senescent cells were induced by DTX as aforementioned, washed with PBS and incubated in fresh medium with Stattic, K1823 and K1836 for 4 h; the lysates were then prepared as aforementioned for proliferating cells. Cell lysates were then heated at 95°C for 5 min and sonicated (3×30 sec at 3 µm amplitude and 40 kHz with 30 sec cooling intervals) using a Diagenode Bioruptor 300 (Diagenode SA). Before separation by SDS-PAGE on 10% gels, 0.01% bromophenol blue was added to the lysates. Equal amounts of protein (35 µg) were loaded per lane. After separation, proteins were electro-transferred onto a nitrocellulose membrane (Bio-Rad Laboratories, Inc.) using semi-dry transfer and were detected using primary antibodies at 4°C for 24 h, followed by incubation with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. GAPDH was used as a loading control.

RNA samples from TC-1 and TRAMP-C2 cell lines were isolated using an RNeasy Plus mini kit (Qiagen Sciences, Inc.) according to the manufacturer's protocol. cDNA was synthesized with random hexamer primers using a High-Capacity cDNA RT kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). The temperature protocol for RT was 42°C for 30 min, 99°C for 5 min and 10°C for 5 min. qPCR was performed using an LC480II system (Roche Applied Science) with the SYBR™ Select Master mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). The samples underwent a denaturation step (95°C for 6 min), followed by 42 amplification cycles (95°C for 30 sec, 60°C for 50 sec and 72°C for 70 sec), a melting step (95°C for 1 min, 65°C for 1 min and 95°C continuous acquisition) and cooling (37°C for 1 min). The relative quantity of cDNA was quantified using the 2^−ΔΔCq method (27). The sequence of the primers used were as follows: β-actin forward (F), 5′-CATTGCTGACAGGATGCAGAAGG-3′ and reverse (R), 5′-TGCTGGAAGGTGGACAGTGAGG-3′; IL-6 F, 5′-GGCCTTCCCTACTTCACAAG-3′ and R, 5′-ATTTCCACGATTTCCCAGAG-3′; GROα F, 5′-ACCCAAACCGAAGTCATAGC-3′ and R, 5′-TCTCCGTTACTTGGGGACAC-3′; and MCP-1 F, 5′-GAAGGAATGGGTCCAGACAT-3′ and R, 5′-ACGGGTCAACTTCACATTCA-3′, all primers were purchased from East Port Praha s.r.o. The final concentration of the primers used was 1 µM. Fold changes in transcript levels were calculated relative to β-actin, which was used as the endogenous reference gene control. The relative expression in the control group was normalized to 1. All samples were run in triplicate.

The protein levels of murine GROα were assessed in the supernatants of proliferating and senescent TC-1 and TRAMP-C2 cells using high-sensitivity ELISA kits (cat. no. DY453; R&D Systems, Inc.). After 4-day induction of senescence, cells were washed twice with PBS to remove DTX in the medium to avoid its possible influence on the results. K1836 was thereafter added for 72 h with repeated supplementation every 24 h. After cell treatment at 37°C, the medium was changed and the cells were cultivated for another 48 h in fresh medium. Experiments were performed according to the manufacturer's protocol and absorbance was quantified using a Thermo Scientific Multiskan EX (Thermo Fisher Scientific, Inc.).

The levels of selected inflammatory factors (IL-6, IL-10, MCP-1, IFN-γ, TNF-α and IL-12) secreted by treated and untreated tumour cells were evaluated in media samples using a Mouse Inflammation Kit (cat. no. 552364; BD Biosciences) according to the manufacturer's protocol. The samples were analysed using a BD FACS Symphony™ flow cytometer (BD Biosciences). Data were analysed using FlowJo 10 software (FlowJo LLC).

Graphs were generated using GraphPad Prism 9 (version 9.4.0; GraphPad Software; Dotmatics). Statistical analyses of graphs were performed using one-way ANOVA followed by Dunnett's post hoc test.

Two murine tumour cell lines, TC-1 and TRAMP-C2, which have constitutively elevated levels of activated pSTAT3, were used. Senescence was induced in these cell lines using 7.5 µM DTX as described previously (7,22). To compare the cytotoxic effects of commercially available STAT3 inhibitor Stattic and its two analogues, compound K1823 and newly synthesized K1836, proliferating and senescent TC-1 and TRAMP-C2 cells were treated with different concentrations of the tested compounds for 24 h and the cytotoxicity was assessed using the MTT assay. Both Stattic and K1823 demonstrated cytotoxic effects on both TC-1 and TRAMP-C2 proliferating cells. K1836 had a markedly lower cytotoxic effect compared with Stattic and K1823, as demonstrated by the IC₅₀ values for particular inhibitors (Fig. 2; Table I). The MTT test generates a cell number estimation by measurement of their metabolic activity, so it does not discriminate between cell death and decreased cell proliferation. Therefore, flow cytometric analysis using PI and Annexin V staining was used to evaluate the early effects of the inhibitors on the cells. Proliferating and senescent cells were treated with 5, 15 and 30 µM Stattic, K1823 and K1836 for 24 h and the proportion of live cells was estimated as the percentage of Annexin V-negative/PI-negative cells (Figs. 3 and S5). Significant cytotoxic effects of both 30 µM Stattic and 30 µM K1823 compounds were demonstrated on proliferating cells, but not on senescent cells (Fig. 3A and B). However, cytotoxicity induced by K1836 was not statistically significant. The cytotoxic effects of Stattic and its two analogues, K1823 and K1836, following treatment of proliferating mouse TRAMP-C2 and human breast adenocarcinoma MDA-MB-231 cells for 24 or 72 h were assessed using the CellTiter-Glo luminescent cell viability assay. The results were similar to those using murine cell lines, as K1836 was the analogue with the lower cytotoxic activity. Longer incubation with the inhibitor decreased the IC₅₀ in both mouse and human cell lines (Table II).

The inhibitory effects of Stattic and the K1823 and K1836 analogues on STAT3 phosphorylation were evaluated using western blotting in proliferating TC-1 and TRAMP-C2 cells, both of which demonstrated constitutive phosphorylation of STAT3 (Fig. 4A and B). Cells were treated with 5, 15 and 30 µM Stattic, K1823 and K1836 for 4 h, and then the protein expression levels of pSTAT3 (Y705), as well as of total STAT3, were assessed. Furthermore, DTX-induced senescent cells (4 days after DTX treatment) were evaluated in the same way. Stattic and its analogues markedly inhibited Y705 STAT3 phosphorylation in both senescent TC-1 (Fig. 4C) and TRAMP-C2 (Fig. 4D) cells in a concentration-dependent manner, similar to the effect demonstrated in proliferating cells. The total STAT3 and pSTAT3 protein expression levels in the control TC-1 sample were constitutive but not stable, and continual changes in STAT3 protein expression levels in the TC-1 cell culture were noted in experiments, possibly due to oscillations in its expression.

How the STAT3 inhibitor K1836, which was only moderately cytotoxic but inhibited STAT3 phosphorylation in senescent cells, was able to affect the induction of senescence by DTX was evaluated. Both tumour cell lines were cultured with 15 µM K1836 for 72 h (with repeated supplementation every 24 h, as it was previously observed that STAT3 inhibition was not permanent and lasted for ~24 h; data not shown). K1836 was added to the cell culture medium either 4 h before adding 7.5 µM DTX (pre-treatment) or after 4 days of exposure to 7.5 µM DTX (post-treatment). There was no significant effect when K1836 was added either before or after DTX supplementation on the cell count or senescence of either the TC-1 (Fig. 5A and B) or TRAMP-C2 (Fig. 5C and D) tumour cell lines. The induction or presence of senescence upon DTX and inhibitors treatment was assessed using SA-β-gal activity in both TC-1 (Fig. 5B) and TRAMP-C2 (Fig. 5D) cell lines.

To analyse the effects of K1836 on the senescent cell phenotype in more detail, SASP modulation was evaluated. Changes in the expression and production of important SASP components upon K1836 treatment of senescent cells were assessed. ELISA and the CBA mouse inflammation kit were used to quantify the levels of selected SASP components (pro-inflammatory cytokines IL-6, GROα, MCP-1, IL-10, IFNγ and IL-12) in the supernatant from senescent cell cultures that were, after senescence induction, cultured for 72 h with 15 µM K1836 and then fresh medium for 48 h, before supernatant collection. Furthermore, the expression of the respective genes was assessed using RT-qPCR in corresponding cell samples. The secretion of IL-6 (CBA; Figs. 6B and E, and S6), GROα (ELISA; Figs. 6A and D, and S6), and MCP-1 (CBA; Figs. 6C and F and S6) was markedly increased in both TC-1 and TRAMP-C2 senescent cells compared with the control and were markedly reduced after the treatment of the senescent cells with K1836. The CBA analysis also demonstrated no changes in IFN-γ, TNF-α, IL-10 and IL-12 levels (data not shown). Corresponding significant reductions in IL-6, GROα, and MCP-1 mRNA expression levels were demonstrated in both TC-1 and TRAMP-C2 senescent cells following treatment with K1836 (Fig. 7).