In the present study, the BON-1 cell line (RRUD: CVCL_3985) (NCL2110P096; DBA Italia s.r.l.) (https:www.cellosaurus.org/CVCL_3985), established from a lymph node metastasis of a human pancreatic carcinoid tumor (27), was used. Cells arrived at passage 1 and were used for additional 6 passages. Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Corning, Inc.), plus glutamine and antibiotics [Penicillin-Streptomycin-L-Glutamine, 100× (+) 29.2 mg/ml L-glutamine; cat. no. 30-009-CI; Merck KGaA] in a humidified atmosphere with 5% CO₂ at 37°C. Cells underwent routine testing to ensure that they were mycoplasm negative. The cationic Ruthenium (Ru)(II) compound containing bisdemethoxycurcumin and the hydrosoluble PTA phosphine ([(cym)Ru(bdcurc)(PTA)]SO3CF3) (where cym=cymene, bdcurc=bisdemethoxycurcumin and PTA=1,3,5-triaza-7-phosphaadamantane) (herein Ru-bdcurc), with the chemical formula: C36H42F3N3O7PRuS and the molecular weight: 849,8 g/mol, was synthesized as previously reported (16,17). The Ru-bdcurc compound was dissolved in DMSO and stored at −20°C before using it at 50 and 100 µM for the indicated times, as previously reported (19). The inhibitor of the antioxidant response Brusatol (cat. no. SML1868; Sigma-Aldrich; Merck KGaA) (35,36) was used at 100 µM for 4 h pre-treatment, as previously reported (37).

Cell viability was measured by Trypan blue (cat. no. 72571; Sigma-Aldrich; Merck KGaA) assay. Subconfluent cells were plated in six-well plates and, the day after, treated with different concentration of Ru-bdcurc for 24 and 48 h or in combinations with a 4 h pre-treatment of NRF2 inhibitor Brusatol (100 nM). After treatments, both floating and adherent cells were collected and stained with Trypan blue. Cell viability of triplicates was assessed by counting blue (dead)/total cells with a Neubauer hemocytometer using light microscopy. For long-term cell survival, cells were plated in 60 mm Petri dishes until subconfluence. Then, cells were mock-treated or treated with Ru-bdcurc (50 or 100 µM) for 16 h. After treatments, cells were washed, trypsinized, counted and equal cell number re-plated in duplicate with fresh medium in 60-mm Petri dishes for determining cell survival. Death-resistant colonies (with >50 cells) were stained with crystal violet (cat. no. 46364; Sigma-Aldrich; Merck KGaA) (diluted 1:2 with the cell culture) 14 days later. Plates underwent scanning and the intensity of the cell staining was quantified by ImageJ software.

Cells were harvested and centrifuged and the resulting pellets were lysed in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 150 mM KCl, 1 mM dithiothreitol and 1% Nonidet P-40) (all from Sigma-Aldrich; Merck KGaA) containing protease inhibitors (CompleteTM, Mini Protease Inhibitor Cocktail; Merck Life Science S.r.l.). Protein concentration was determined by the Bio-Rad protein assay kit (cat. no. 5000001; Bio-Rad Laboratories, Inc.), a simple colorimetric assay for measuring total protein concentration based on the Bradford dye-binding method. Proteins were separated by loading 10–30 µg of total cell lysates on denaturing 8–15% SDS-PAGE (polyacrylamide gel electrophoresis) gels (Bio-Rad Laboratories, Inc.), following semidry blotting to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Merck KGaA). Unspecific signals were blocked by incubating the membranes in Tris-buffered saline containing 0.1% Tween 20 (TBS) and 3% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Membranes were then probed with the primary antibodies and subsequently with the following secondary antibodies: Goat Anti-Mouse IgG (1:10,000; cat. no. 1706516) and Goat Anti-Rabbit IgG (1:10,000; cat. no. 1706515; both from Bio-Rad Laboratories, Inc.). The enzymatic signal was visualized by chemiluminescence (ECL Detection system; Amersham; Cytiva). The following antibodies were used: Mouse monoclonal anti-p62/SQSTM1 (D-3; 1:1,000; cat. no. sc-28359), mouse monoclonal anti-catalase (H-9; 1:1,000; cat. no. sc-271803), mouse monoclonal anti-p53 (DO-1; 1:1,000; cat. no. sc-126), mouse monoclonal anti-Bcl-2 (1:1,000; cat. no. sc-509) and mouse monoclonal anti-Mcl1 (G-7; 1:1,000; cat. no. sc-74437) (all from Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-NRF2 (1:1,000; cat. no. ab62352; Abcam), mouse monoclonal anti-phospho-Histone H2AX (Ser139 clone JBW301; 1:1,000; cat. no. 05-636; Sigma-Aldrich; Merck KGaA), rabbit polyclonal anti-phospho-4E-BP1 (Thr37/46; 1:200; ca.t no. 2855), rabbit polyclonal anti-4E-BP1 (1:200; cat. no. 9452; both from Cell Signaling Technology, Inc.), mouse monoclonal anti-poly(ADP-ribose) polymerase (PARP, cleavage site-214-215; 1:1,000; cat. no. AB3565; Sigma-Aldrich; Merck KGaA). Mouse monoclonal β-actin (Ab-1; 1:10,000; (cat. no. CP01; Calbiochem; Merck KGaA), was used as protein loading control.

Densitometry was performed on ECL results with ImageJ software (1.47 version; National Institutes of Health) which was downloaded from the NIH website (http://imagej,nih.gov/ij) and the relative band intensity was normalized to β-actin signals and plotted as protein expression/β-actin ratio.

Cells were plated at subconfluency in 35-mm Petri dishes and, the day after plating, were transfected with the Nrf2 small interference (si)RNA (cat. no. sc-37030) or control siRNA (cat. no. sc-37007) (both from Santa Cruz Biotechnology, Inc.; the siRNA sequences are not available) using LipofectaminePLus reagent (cat. no. 11514015; Thermo Fisher Scientific, Inc.) as previously reported (18). The concentration used was 10 nmol. For p53 knockdown, cells were transfected with sip53 plasmid (sip53) or an empty vector (si-ctr) (38,39) using LipofectaminePLus reagent according to the manufacturer's instructions. A total of 24 h after transfection, cells were trypsinized and replated for the indicated experiments.

Total RNA extraction was performed by using TRIzol Reagent (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. cDNA was synthesized by using MuLV reverse transcriptase kit according to the manufacturer's instructions (Applied Biosystems; Thermo Fisher Scientific, Inc.). Semiquantitative RT-PCR was carried out with 2 µl cDNA reaction and genes specific oligonucleotides under conditions of linear amplification. by using Hot-Master Taq polymerase (Thermo Fisher Scientific, Inc.). Primer sequences specific to the target genes were as follows: HO-1 forward, 5′AAGATTGCCCAGAAAGCCCTGGAC-3′ and reverse, 5′-AACTGTCGCCACCAGAAAGCTGAG-3′ (40) (annealing temperature: 58°C for 30 cycles); p62 forward, 5′-CTGCCCAGACTACGACTTGTGT-3′ and reverse, 5′-TCAACTTCAATGCCCAGAGG-3′ (19,40) (annealing temperature: 58°C for 28 cycles); NRF2 forward, 5′-TCCATTCCTGAGTTACAGTGTCT-3′ and reverse, 5′-TGGCTTCTGGACTTGGAACC-3′ (18,40,41) (annealing temperature: 58°C for 30 cycles); MDR1 forward, 5′-AACGGAAGCCAGAACATTCC-3′ and reverse, 5′-AGGCTTCCTGTGGCAAAGAG-3′ (42,43) (annealing temperature: 60°C for 29 cycles); 28S forward, 5′-GTTCACCCACTAATAGGGAACGTGA-3′ and reverse, 5′-GGATTCTGACTTAGAGGCGTTCAGT-3′ (39,40,44,45) (annealing temperature: 58°C for 15 cycles); Bcl-2 forward, 5′-AGGATTGTGGCCTTCTTTGAG-3′ and reverse, 5′-GAGACAGCCAGGAGAAATCAAA-3′ (46) (annealing temperature: 58°C for 30 cycles); and NOXA forward, 5′-AGGACTGTTCGTGTTCAGCTC-3′ and reverse 5′-GTCCACCTCCTGAGAAAACTC-3′ (47) (annealing temperature: 55°C for 28 cycles). The denaturation and extension temperatures were, respectively 98 and 72°C for all the mRNA amplifications. PCR products were run on a 2% agarose gel and visualized with GelRed Nucleic Acid gel stain (Biotium, Inc.). The housekeeping 28S gene, used as internal standard, was amplified from the same cDNA reaction mixture. Densitometric analysis was applied to quantify mRNA levels compared with 28S control gene expression.

The results are expressed as the mean ± standard deviation (SD) of at least three independent experiments and statistical analyses were performed using GraphPad Prism® software (Version 9.0.0; Dotmatics). The unpaired two-tailed Student t-test (for data containing two groups) and the nonparametric 1-way analysis of variance (ANOVA) followed by Tukey's HSD test (for multiple comparisons tests) were used to demonstrate statistical significance. A difference was considered statistically significant when P≤0.05.

BON-1 cells were treated with two concentrations of Ru-bdcurc compound that were recently demonstrated by the authors to have different cytotoxic activity against colon cancer cells (19). The results revealed a dose- and time-dependent cell death, as indicated by the trypan blue assay (Fig. 1A). Cell death was also evidenced microscopically where distinct signs of cell shrinkage were observed, in particular at 100 µM dose of Ru-bdcurc (Fig. 1B). The EC50 for the Ru-bdcurc compound was 100 µM (AAT Bioquest, Inc; http://www.aatbio.com/tools/ic50-calculator). Then the long-term survival was analyzed by colony formation assay. The results of the densitometric analysis demonstrated that 100 µM dose of Ru-bdcurc significantly reduced BON-1 cell survival, compared with 50 µM dose (Fig. 1C). At the biochemical level, the occurrence of an apoptotic cell death, as indicated by the cleaved fragment of PARP (clPARP) and by the increased clPARP/PARP ratio, was evident only with the 100 µM dose treatment, compared with 50 µM dose (Fig. 1D). The apoptotic cell death associated with the appearance of the phosphorylated form of H2AX (γH2AX) (Fig. 1D), a marker of DNA damage and apoptosis (48). Then the phosphorylation of 4EBP1 (p4E-BP1), a mTOR target whose activation can sustain cancer cell survival and predict poor prognosis (49,50), was investigated. In agreement, mTOR pathway has been found dysregulated in GEP-NET and involved in tumor development (51). As shown in Fig. 1D, p4E-BP1 was induced by 50 µM dose, on the other hand, p4E-BP1 was impaired by 100 µM dose of Ru-bdcurc. This latter result associated with the greater induction of cell death that was observed by using 100 µM dose of Ru-bdcurc, compared with 50 µM dose, strengthening the antiapoptotic role of p4E-BP1. Collectively, these results indicated that 100 µM dose of Ru-bdcurc induced BON-1 cell death while 50 µM dose activated survival pathways that potentially reduced the compound cytotoxic effects.

Several lines of evidence suggest the important role of NRF2 in the chemoresistance of different types of cancer cells (22), as also assessed by the authors' recent studies using curcumin compounds (18,19). However, to the best of the authors' knowledge, NRF2 has never been evaluated in BON-1 cells. Therefore, the NRF2 pathway in BON-1 cells in response to the Ru-bdcurc treatment was next assessed. To this aim, the two doses of the compound, that were revealed to have different outcome in terms of cell death as aforementioned, were used. The experimental data demonstrated that 50 µM dose of Ru-bdcurc significantly increased the levels of NRF2 protein, compared with the 100 µM dose, and induced the expression of its targets such as catalase and p62 (Fig. 2A). To evaluate if the NRF2 induction was at the transcriptional or post-transcriptional level, mRNA analysis was performed. It was identified that 50 µM dose of Ru-bdcurc did not induce NRF2 gene expression (Fig. 2B), suggesting rather stabilization of NRF2 at the protein level. In addition, only the 50 µM dose increased the mRNA expression of the NRF2 targets HO-1 and p62 (Fig. 2B), indicative of NRF2 transcriptional activation, in this setting. Thus, the increased p62 gene expression was in accordance with the finding that p62 is a NRF2 transcriptional target (23–25). Interestingly, it has been reported that p62 activates pro-survival pathways including mTOR (52), in agreement with the aforementioned results in Fig. 1 and with the cross-talk among oncogenic pathways such as p62/mTOR/NRF2 to increase tumor development and resistance to therapies (30). In agreement with the different extent of cell death in response to different doses of Ru-bdcurc, the results showed that only 50 µM dose of Ru-bdcurc increased the antiapoptotic Bcl-2 protein levels while the 100 µM dose significantly reduced the levels of Mcl1 protein (Fig. 2A), a pro-survival member of the Bcl-2 family (53). Moreover, 50 µM dose of Ru-bdcurc treatment, compared with 100 µM dose, increased the endogenous p53 protein level (Fig. 2A), in agreement with the paradigm of NRF2/mutp53 interplay to sustain their oncogenic activities (41,54). The endogenous p53 is reported to be dysfunctional in BON-1 cells and inhibit apoptosis (28). In agreement, the present results revealed that 50 µM dose of Ru-bdcurc, compared with 100 µM dose, induced the expression of multidrug resistant gene 1 (MDR1), a target of some mutant p53 proteins (55), as well as of the antiapoptotic gene Bcl-2 (Fig. 2B), which associated with a potential oncogenic activity of the endogenous dysfunctional p53 in BON-1 cells. On the other hand, 100 µM dose of Ru-bdcurc induced the expression of Noxa (Fig. 2B), a pro-apoptotic gene induced by p53 family members (56), that has been shown to inhibit the antiapoptotic Mcl1 protein (57). This latter result is in consistency with the reduction of Mcl1 protein levels in Fig. 2A and with induction of the apoptotic cell death (Fig. 1A). Taken together, these findings indicated that the Ru-bdcurc treatment was able to induce pro-apoptotic (PARP cleavage, Fig. 1) or cell death-resistant pathways (NRF2-induced targets, mTOR target 4E-BP1, Bcl-2 and dysfunctional p53; Fig. 2) according to the low (50 µM) or high (100 µM) dose used.

Then, to evaluate the biological role of NRF2 in this setting, it was attempted to inhibit it by pharmacologic or genetic means. NRF2 pharmacologic inhibitor Brusatol (35,36) was used before exposing BON-1 cells to 50 µM of Ru-bdcurc. The results demonstrated that the Ru-bdcurc/Brusatol combination increased BON-1 cell death (Fig. 3A, upper panel), compared with the 50 µM dose Ru-bdcurc alone. At the biochemical level, the Ru-bdcurc/Brusatol combination counteracted the Ru-bdcurc-induced upregulation of NRF2 as well as of its target p62 (Fig. 3A, lower panels); in addition, the Ru-bdcurc/Brusatol combination significantly increased the expression of γH2AX compared with the 50 µM dose of Ru-bdcurc alone (Fig. 3A, lower panels). Similarly, knocking down NRF2 by specific siRNA (Fig. 3B) increased the cell death induced by 50 µM dose of Ru-bdcurc compound (Fig. 3C, left panels). At the biochemical level, the NRF2 silencing reduced catalase and p62 expression, as well as the p53 levels that were induced in response to 50 µM dose of Ru-bdcurc (Fig. 3C, right panels), suggesting that an interplay between NRF2 and dysfunctional p53 exists in BON-1 cells. In addition, the Ru-bdcurc/Brusatol combination significantly increased the expression of γH2AX compared with the 50 µM dose of Ru-bdcurc alone (Fig. 3C, lower panels). Collectively, these results suggested that inhibiting the NRF2 pathway, induced by the lower dose of Ru-bdcurc, increased the cytotoxic effect of Ru-bdcurc compound.

Then, to evaluate the role of p53 in BON-1 cell response to the Ru-bdcurc compound and its interplay with NRF2, it was attempted to inhibit it by specific siRNA (38,39). The results demonstrated that knocking down p53 (Fig. 4A) reduced the Ru-bdcurc-induced upregulation of NRF2 and of its targets p62 and catalase (Fig. 4B). At the biological level, knocking down p53 increased the cell death induced by 50 µM dose of Ru-bdcurc (Fig. 4C). Taken together, these results indicated that a dysfunctional endogenous p53 in BON-1 contributed to cellular resistance to Ru-bdcurc cytotoxicity, likely in an interplay with NRF2.