The importance of epigenetic changes as a measurable endpoint in nanotoxicological studies is getting more and more appreciated. In the present work, we analyzed the epigenetic effects induced by citrate- and PEG-coated 20 nm silver nanoparticles (AgNPs) in a model consisting of 4T1 breast cancer tumors in mice. Animals were administered with AgNPs intragastrically (1 mg/kg b.w. daily—total dose 14 mg/kg b.w.) or intravenously (administration twice with 1 mg/kg b.w.—total dose 2 mg/kg b.w.). We observed a significant decrease in 5-methylcytosine (5-mC) level in tumors from mice treated with citrate-coated AgNPs regardless of the route of administration. For PEG-coated AgNPs, a significant decrease in DNA methylation was observed only after intravenous administration. Moreover, treatment of 4T1 tumor-bearing mice with AgNPs decreased histone H3 methylation in tumor tissue. This effect was the most pronounced for PEG-coated AgNPs administered intravenously. No changes in histone H3 Lys9 acetylation were observed. The decrease in methylation of DNA and histone H3 was accompanied by changes in expression of genes encoding chromatin-modifying enzymes (Setd4, Setdb1, Smyd3, Suv39h1, Suv420h1, Whsc1, Kdm1a, Kdm5b, Esco2, Hat1, Myst3, Hdac5, Dnmt1, Ube2b, and Usp22) and genes related to carcinogenesis (Akt1, Brca1, Brca2, Mlh1, Myb, Ccnd1, and Src). The significance of the observed changes and the mechanisms responsible for their development are unclear, and more research in this area is warranted. Nevertheless, the present work points to the epigenetic effects as an important level of interaction between nanomaterials and biological systems, which should always be taken into consideration during analysis of the biological activity of nanomaterials and development of nanopharmaceuticals.

The potential anticancer activity of different silver nanoformulations is increasingly recognized. In the present work, we use the model of 4T1 tumor in BALB/ccmdb immunocompetent mice to analyze the impact of citrate- and PEG-coated silver nanoparticles (AgNPs) on the development and metastatic potential of breast cancer. One group of mice was intragastrically administered with 1 mg/kg body weight (b.w.) of AgNPs daily from day 1 to day 14 after cancer cells implantation (total dose 14 mg/kg b.w.). The second group was intravenously administered twice with 1 or 5 mg/kg b.w. of AgNPs. A tendency for lowering tumor volume on day 21 (mean volumes 491.31, 428.88, and 386.83 mm³ for control, AgNPs-PEG, and AgNPs-citrate, respectively) and day 26 (mean volumes 903.20, 764.27, and 672.62 mm³ for control, AgNPs-PEG, and AgNPs-citrate, respectively) has been observed in mice treated intragastrically, but the effect did not reach the level of statistical significance. Interestingly, in mice treated intragastrically with citrate-coated AgNPs, the number of lung metastases was significantly lower, as compared to control mice (the mean number of metastases 18.89, 14.90, and 8.03 for control, AgNPs-PEG, and AgNPs-citrate, respectively). No effect of AgNPs treatment on the number of lung metastases was observed after intravenous administration (the mean number of metastases 12.44, 9.86, 12.88, 11.05, and 10.5 for control, AgNPs-PEG 1 mg/kg, AgNPs-PEG 5 mg/kg, AgNPs-citrate 1 mg/kg, and AgNPs-citrate 5 mg/kg, respectively). Surprisingly, inhibition of metastasis was not accompanied by changes in the expression of genes associated with epithelial–mesenchymal transition. Instead, changes in the expression of inflammation-related genes were observed. The presented results support the antitumor activity of AgNPs in vivo, but the effect was limited to the inhibition of metastasis. Moreover, our results clearly point to the importance of AgNPs coating and route of administration for its anticancer activity. Finally, our study supports the previous findings that antitumor AgNPs activity may depend on the activation of the immune system and not on the direct action of AgNPs on cancer cells.

Radiotherapy is a cornerstone of cancer treatment, but tumor hypoxia and resistance to radiation remain significant challenges. Vascular normalization has emerged as a strategy to improve oxygenation and enhance therapeutic outcomes. In this study, we examine the radiosensitization potential of vascular normalization using metformin, a widely used anti-diabetic drug, and oxygen microbubbles (OMBs). We investigated the synergistic action of metformin and OMBs and the impact of this therapeutic combination on the vasculature, oxygenation, invasiveness, and radiosensitivity of murine 4T1 breast cancer. We employed in vivo Doppler ultrasonographic imaging for vasculature analysis, electron paramagnetic resonance oximetry, and immunohistochemical assessment of microvessels, perfusion, and invasiveness markers. Our findings demonstrate that both two-week metformin therapy and oxygen microbubble treatment normalize abnormal cancer vasculature. The combination of metformin and OMB yielded more pronounced and sustained effects than either treatment alone. The investigated therapy protocols led to nearly twice the radiosensitivity of 4T1 tumors; however, no significant differences in radiosensitivity were observed between the various treatment groups. Despite these improvements, resistance to treatment inevitably emerged, leading to the recurrence of hypoxia and an increased incidence of metastasis.

Background: Glioblastoma multiforme (GBM) is the most common highly aggressive, primary malignant brain tumor in adults. Current experimental strategies include photodynamic therapy (PDT) and new drug delivery technologies such as nanoparticles, which could play a key role in the treatment, diagnosis, and imaging of brain tumors. Objectives: The purpose of this study was to test the efficacy of PDT using AGuIX-TPP, a polysiloxane-based nanoparticle (AGuIX) that contains TPP (5,10,15,20-tetraphenyl-21H,23H-porphine), in biological models of glioblastoma multiforme and to investigate the vascular mechanisms of action at multiple complexity levels. Methods: PDT effects were studied in monolayer and spheroid cell culture, as well as tumors in chicken chorioallantoic membranes (CAMs) and in mice were studied. Results: Treatment was effective in both endothelial ECRF and glioma U87 cells, as well as in the inhibition of growth of the glioma spheroids. PDT using AGuIX-TPP inhibited U87 tumors growing in CAM and destroyed their vascularization. The U87 tumors were also grown in nude mice. Their vascular network, as well as oxygen partial pressure, were assessed using ultrasound and EPR oximetry. The treatment damaged tumor vessels and slightly decreased oxygen levels. Conclusions: PDT with AGuIX-TPP was effective against glioma cells, spheroids, and tumors; however, in mice, its efficacy appeared to be strongly related to the presence of blood vessels in the tumor before the treatment.

Gold nanoparticles (AuNPs) are foreseen as a promising tool in nanomedicine, both as drug carriers and radiosensitizers. They have been also proposed as a potential anticancer drug due to the anti-angiogenic effect in tumor tissue. In this work we investigated the effect of citrate-coated AuNPs of nominal diameter 20 nm on the growth and metastatic potential of 4T1 cells originated from a mouse mammary gland tumor inoculated into the mammary fat pad of Balb/ccmdb mice. To evaluate whether AuNPs can prevent the tumor growth, one group of inoculated mice was intragastrically (i.g.) administered with 1 mg/kg of AuNPs daily from day 1 to day 14 after cancer cell implantation. To evaluate whether AuNPs can attenuate the tumor growth, the second group was intravenously (i.v.) administered with 1 or 5 mg/kg of AuNPs, twice on day 5 and day 14 after inoculation. We did not observe any anticancer activity of i.v. nor i.g. administered AuNPs, as they did not affect neither the primary tumor growth rate nor the number of lung metastases. Unexpectedly, both AuNP treatment regimens caused a marked vasodilating effect in the tumor tissue. As no change of potential angiogenic genes (Fgf2, Vegfa) nor inducible nitric oxygenase (Nos2) was observed, we proposed that the vasodilation was caused by AuNP-dependent decomposition of nitrosothiols and direct release of nitric oxide in the tumor tissue.

Premelanosomal protein (Pmel, also known as Pmel17) is the major component of melanosomal fibrils and plays a key role in melanin polymerization, making it an important factor in melanogenesis. We investigated how the absence of Pmel affects the properties of B16F10 melanoma cells. Pmel-knockout B16F10 cells were generated using CRISPR/Cas9-mediated genome editing. A viability assay revealed no significant differences between wild-type (WT) and Pmel-knockout (KO) sublines; however, melanosome maturation was impaired. In Pmel KO cells, the cell cycle was disrupted, and higher levels of reactive oxygen species (ROS) were observed compared with WT cells. Moreover, the migration capacity and tube formation of melanoma cells were increased. Tumors derived from Pmel KO cells exhibited unchanged growth kinetics but reduced melanin content, along with enhanced vascularization and oxygenation. Thus, knockout of the Pmel17 gene in melanoma cells alters pigmentation, vascularization, and oxygenation of tumors. These parameters are crucial for both tumor progression and therapeutic response.