Titania Nanosheets Generates Peroxynitrite for S-Nitrosylation and Enhanced p53 Function in Lung Cancer Cells

Background: Metal oxide nanomaterials are increasingly being exploited in cancer therapy thanks to their unique properties, which can enhance the efficacy of current cancer therapies. However, the nanotoxicity and mechanism of Ti 0.8 O 2 nanosheets for specific site-targeting strategies in NSCLC have not yet been investigated. Methods: The effects of Ti 0.8 O 2 nanosheets on cytotoxicity in NSCLC cells and normal cells were examined. The apoptosis characteristics, including condensed and fragmented nuclei, as assessed by positive staining with annexin V. The cellular uptake of the nanosheets and the induction of stress fiber were assessed via transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses, respectively. We also evaluated the expression of protein in death mechanism to identify the molecular mechanisms behind the toxicity of these cells. We investigated the relationship between S -nitrosylation and the increase in p53 stability by molecular dynamics. Results: Ti 0.8 O 2 nanosheets caused cytotoxicity in several lung cancer cells, but not in normal cells. The nanosheets could enter lung cancer cells and exert an apoptosis induction. Results for protein analysis further indicated the activation of p53, increased Bax, decreased Bcl-2 and Mcl-1, and activation of caspase-3. The nanosheets also exhibited a substantial apoptosis effect in drug-resistant metastatic primary lung cancer cells, and it was found that the potency of the nanosheets was dramatically higher than that of cisplatin and etoposide. In terms of their mechanism of action, we found that the mode of apoptosis induction was through the generation of cellular ONOO − mediated the S -nitrosylation of p53 at C182. Molecular dynamics analysis further showed that the S -nitrosylation of one C182 stabilized the p53 dimer. Consequently, this nitrosylation of the protein led to an upregulation of p53 through its stabilization. Conclusions: Taking all the evidence together, we provided information on the apoptosis induction effect of the nanosheets through a molecular mechanism involving reactive nitrogen species, which affects the protein stability; thus emphasizing the novel mechanism of action of nanomaterials for cancer therapy. patient-derived malignant cancer cells were isolated from pleural effusions of recurrent advanced stage non-small cell lung cancer patients who had been The was (IRB 365/62) and was obtained informed consents from all contributors. Primary cancer cells were collected from pleural effusion (500-1,000 mL) through thoracentesis. The collected samples were centrifuged at 300 g for 10 min, at 4˚C and the cells were resuspended in RPMI medium with 10% FBS, 2 L glutamine, and 100 units/ml of each of penicillin and streptomycin. After culturing for 10-15 passages, they were characterized as the patient-derived primary cancer cell lines (PM-4, ELC09, ELC12, ELC16, ELC17, and ELC20). patients. A-F Effect of the Ti0.8O2 nanosheets on the cell viability of malignant pleural effusion for 24 h using the MTT assay to determine the IC50 values. G Percentages of cell viability were determined using the MTT assay. H-M Morphology of apoptotic nuclei stained with Hoechst 33342 dye and propidium iodide in cells treated with Ti0.8O2 nanosheets, cisplatin and etoposide were determined by visualization under a uorescence microscope; the percentages of nuclear fragments and PI positive cells were calculated. Data are shown as the mean SD (n 3). P versus non-treated

Under SEM morphological analysis, it was seen that for H460 cells, the morphology of 150 the cancer cells gradually changed, including the formation of stress fibers, when 151 treated with Ti0.8O2 nanosheets at concentrations of 1-10 μg/mL (Fig. 3A). Moreover,

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Ti 0.8 O 2 nanosheets modulate apoptosis-related proteins in H460 and A549 cells 156 In order to investigate the mechanism of Ti0.8O2 nanosheets-induced apoptosis, the 157 apoptotic-related proteins were determined by Western blot analysis. A549 and H460 158 cells were treated with 0-10 μg/mL Ti0.8O2 nanosheets, and then the pro-and anti-apoptotic proteins related to mitochondria-mediated apoptosis were evaluated. The 160 results showed that the Ti0.8O2 nanosheets increased the pro-apoptotic protein Bax,

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whereas the anti-apoptotic proteins Mcl-1 and Bcl-2 were downregulated in the cells 162 treated with the Ti0.8O2 nanosheets. In addition, pro-caspase3 was decreased in a 163 concentration-dependent manner. Moreover, p53 was found to be activated in 164 response to the treatment with the Ti0.8O2 nanosheets ( Fig. 3C and D). Taken together,

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it can be concluded that the Ti0.8O2 nanosheets mediated the apoptosis of lung cancer 166 cells by increasing the pro-apoptotic proteins, which led to cell death by the 167 mitochondria-dependent pathway. analysis by MTT assay. The Ti0.8O2 nanosheets could be considered nontoxic at doses 183 lower than 1 μg/mL, while a concentration of more than 10 μg/mL caused a 184 significant decrease in the cell viability of the cells (Fig. 4A-F); whereas, the standard 185 drugs showed a slightly decreased cell viability from 0.5 μg/mL to 100 μg/mL, while 186 doses of more than 20 μg/mL cisplatin and etoposide were considered toxic. Data 187 analysis showed that the IC50 of the Ti0.8O2 nanosheets was lower than 10 μg/mL at 24 188 h, which was significantly lower than for cisplatin and etoposide ( Fig. 4M and N). The 189 results showed that the Ti0.8O2 nanosheets reduced cell viability in a concentration-   Ti0.8O2 nanosheets. We detected a decrease in the ROS level in all the cell lines treated 211 with NAC and GSH ( Fig. 5A and B), but the cell viability of the cancer cells could not be 212 reversed by the pretreatment with NAC or GSH ( Fig. 5C and D). These results

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suggested that the Ti0.8O2 nanosheets induce cytotoxicity in cancer cell lines but did 214 not do this via the generation of ROS. Next, we investigated the specific ROS products 215 using a DHE (dihydroethidium) fluorescent probe for the detection of ROS generation,

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and specifically for the detection of superoxide anions. The results showed that the 217 Ti0.8O2 nanosheets had a significant effect on the superoxide anions in H460 cells when they were treated with Ti0.8O2 nanosheets in a concentration-dependent 219 manner (Fig. 5E); while the Ti0.8O2 nanosheets had only a slight effect on superoxide 220 anion generation in A549 cells (Fig. 5E). In addition, we also investigated the 221 generation of hydroxyl radicals using the HPF (hydroxyphenyl fluorescein) 222 fluorescent probe in both cell lines. The results showed that the Ti0.8O2 nanosheets 223 significantly generated hydroxyl radicals in both cell lines compared with the non-224 treated cells (Fig. 5F). According to our obtained data, the pretreatment of cancer cell 225 lines with a potent antioxidant for 1 h could not inhibit H2O2 damage, while the Ti0.8O2 226 nanosheets generated superoxide anion hydroxyl radicals in both cell lines.

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Nitric oxide plays a role in apoptosis regulation through its ability to modulate ROS.

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The cytotoxic capacity of nitric oxide has been confirmed in numerous systems using 231 diverse cell targets. In many circumstances, the cytotoxicity is the result of the   (Fig. 6F). Then, we observed whether increased peroxynitrite was required for cell 246 apoptosis induced by Ti0.8O2 nanosheets. The results showed that the co-treatment 247 with these inhibitors was able to inhibit apoptosis cell death, as shown in Figure 6E.

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To compare the Ti0.8O2 nanosheets stability between Ti0.8O2 nanosheets -treated cells 284 and the control cells, the cycloheximide (CHX) chasing assay was used followed by

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Ubiquitin-proteasome degradation has been shown to influence protein turnover.

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Thus, MG132, a potent proteasome inhibitor, was used to prove that this increase in 297 p53 stability was through proteasomal degradation of the protein by the Ti0.8O2

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This observation suggested that our simulation model was highly stable. Therefore, 324 the equilibrated 100 MD snapshots extracted from the last 20 ns were used for further 325 analysis in terms of the calculation.

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The was then calculated to verify the crucial amino acids involved in 327 protein binding at the interface region of each monomer. The total contributing 328 energy from each amino acid for the protein-protein complex is shown in Figure 9C  could appropriately disperse into H460 cells more easily than in DP cells (Fig. 3B)

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Another previous study suggested that the effect of nanosilver on apoptosis was via the regulation of p53 tetramerization. Our results showed that when the cells were 440 exposed to the nanosheets, the intracellular level of peroxynitrite was highly 441 upregulated (Fig. 6). Concomitantly, increased p53 was detected ( Fig. 7A-C) with the 442 decrease in p53-ubiquitin complex ( Fig. 8D and E), implying that the upregulation of 443 p53 occurs as a result of preventing its degradation process.

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Peroxynitrite is considered an important biological inducer via its direct

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In conclusion, our present study, for the first time provides information on the effect 488 of Ti0.8O2 nanosheets induce apoptosis through a molecular mechanism involving 489 peroxynitrite generation. After treatment with Ti0.8O2 nanosheets, it may directly 490 control p53 by S-nitrosylation to stabilize the tetrameric structure of this protein.

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This reflected that the S-nitrosylated at C182 of p53 resulted in a higher stability of 492 the tetrameric protein-protein complex compared to the native p53. Therefore, the

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The absorption characteristics of the nanosheets colloid was measured using a 510 T90+ UV/VIS spectrometer (PG Instruments). The "size" of the nanosheets (i.e., the

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To confirm that Ti0.8O2 nanosheets were uptake by cancer cell and/or normal cell,

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Finally, protein bands were detected using an enhancement chemiluminescence 609 substrate (Supersignal West Pico; Pierce, Rockford, IL, USA) and exposed to film.

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The treated cells were collected and lysed with RIPA lysis buffer containing the perform Western blot analysis for detecting the ubiquitinated p53 protein.

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Computational method

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The X-ray structure of tetrameric p53 core domain was taken from the protein data   Peroxynitrite-potentiated cell apoptosis through the p53 protein was measured by Western blot analysis. C Blots were reprobed with β-actin to confirm the equal loading of samples. The relative protein levels were calculated by densitometry. Data are shown as the mean ± SD (n = 3). * P < 0.05 versus non-treated control. D, E The expressions of p53 and P-p53 were analyzed by immunofluorescence staining in A549 and H460 cells.

Fig. 8
Ti 0.8 O 2 nanosheets increased p53 stability but not through the p53 proteasomal degradation. A The half-life of p53 was confirmed using the cycloheximine (CHX) chasing assay. H460 and A549 cell lines were treated with 50 µg/ml of CHX with or without 10 µg/ml Ti 0.8 O 2 nanosheets as indicated by the time in minutes. Western blot analysis was performed to evaluate the p53 protein level. B The relative p53 protein levels were calculated and compared with the non-treatment control at 0 min. C The half-lives of the p53 protein of H460 and A549 cells were calculated. D H460 and A549 cells were pretreated with 10 µM MG132 for 30 min followed by treatment with 10 µg/ml Ti 0.8 O 2 nanosheets for 60 min. The protein lysates were collected and incubated with a mixture of beads and p53 primary antibodies to pull out the protein of interest. Then, the ubiquitinated protein levels were measured by Western blot analysis. E Ub-p53 levels were quantified by densitometry. The statistical calculation was compiled with repeated measured one-way ANOVA with Schefft's post-hoc test for individual comparisons and the t-test for two group comparisons. The relative to control protein levels are   Ti0.8O2 nanosheets induced intracellular ROS in H460 and A549 cells. A, B The effect of Ti0.8O2 nanosheets (0-10 g/mL) on intracellular ROS induction at 24 h in H460 and A549 cells was determined by ow cytometry with the uorescent probe DCF (10 µM). Cells were treated with Ti0.8O2 nanosheets (0-10 µg/mL) alone for 24 h or with the pretreatment of 2.5 mM NAC and 2.5 mM GSH. C, D Effect of Ti0.8O2 nanosheets on cell viability in H460 and A549 cells at 24 h with the pretreatment of 2.5 mM NAC or 2.5 mM GSH was determined by the MTT assay. Data are shown as the mean ± SD (n = 3). E The effect of Ti0.8O2 nanosheets (0-10 µg/mL) on superoxide anion induction at 24 h in H460 and A549 cells was determined by ow cytometry with the uorescent probe DHE (10 µM). F The effect of Ti0.8O2 nanosheets (0-10 µg/mL) on hydroxyl radical induction at 24 h in H460 and A549 cells was determined by ow cytometry with the uorescent probe HPF (10 µM). viability. Cells were treated with Ti0.8O2 nanosheets (10 µg/mL) in the presence or absence of pretreatment with a NO scavenger (PTIO) (100 µM) or pretreatment with a superoxide inhibitor (MnTBAP) (50 µM) for 24 h by MTT assay. E Apoptotic and necrotic cells were determined using annexin V-FITC/PI staining with ow cytometry. F Cellular NO level stained with DAF-FM DA in cells treated with Ti0.8O2 nanosheets (10 µg/mL) or pretreated with PTIO (100 µM) or pretreated with MnTBAP (50 µM) were determined by visualization under a uorescence microscope.

Figure 7
Ti0.8O2 nanosheets associated with apoptosis in A549 and H460 cells via p53 upregulation. A, B Peroxynitrite-potentiated cell apoptosis through the p53 protein was measured by Western blot analysis. C Blots were reprobed with β-actin to con rm the equal loading of samples. The relative protein levels were calculated by densitometry. Data are shown as the mean ± SD (n = 3). * P < 0.05 versus non-treated control. D, E The expressions of p53 and P-p53 were analyzed by immuno uorescence staining in A549 and H460 cells.

Figure 8
Ti0.8O2 nanosheets increased p53 stability but not through the p53 proteasomal degradation. A The halflife of p53 was con rmed using the cycloheximine (CHX) chasing assay. H460 and A549 cell lines were treated with 50 µg/ml of CHX with or without 10 µg/ml Ti0.8O2 nanosheets as indicated by the time in minutes. Western blot analysis was performed to evaluate the p53 protein level. B The relative p53 protein levels were calculated and compared with the non-treatment control at 0 min. C The half-lives of the p53 protein of H460 and A549 cells were calculated. D H460 and A549 cells were pretreated with 10 µM MG132 for 30 min followed by treatment with 10 µg/ml Ti0.8O2 nanosheets for 60 min. The protein lysates were collected and incubated with a mixture of beads and p53 primary antibodies to pull out the protein of interest. Then, the ubiquitinated protein levels were measured by Western blot analysis. E Ub-p53 levels were quanti ed by densitometry. The statistical calculation was compiled with repeated measured one-way ANOVA with Schefft's post-hoc test for individual comparisons and the t-test for two group comparisons. The relative to control protein levels are reported. (n = 3) (*p < 0.05, **p < 0.01 compared with the untreated control at 0 min and #< 0.05, ## p < 0.01 compared with the untreated control at the same time).

Figure 9
S-nitrosylation in the regulation of p53 stability. A Three-dimensional (3D) structure of the tetrameric p53 core domain without DNA bound (PDB ID: 3KMD). B Time evolution of the total number of intermolecular hydrogen bonds formed between each monomer of the p53 core domain and its adjacent monomer. C The plot of (kcal/mol) of the p53 tetramer for the native form (top) and the C182 S-nitrosylation (bottom) system. D The representative 3D structure taken from the last MD snapshot of the S-nitrosylation system, with hydrogen bonds and electrostatic interactions represented by black dashed lines.

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