Adenosine triphosphate (ATP) is one of the most important molecules of life, present both inside the cells and extracellularly. It is an essential building block for nucleic acids biosynthesis and crucial intracellular energy storage. However, one of the most interesting functions of ATP is the role of a signaling molecule. Numerous studies indicate the involvement of ATP-dependent pathways in maintaining the proper functioning of individual tissues and organs. Herein, the latest data indicating the ATP function in the network of intra- and extracellular signaling pathways including purinergic signaling, MAP kinase pathway, mTOR and calcium signaling are collected. The main ATP-dependent processes maintaining the proper functioning of the nervous, cardiovascular and immune systems, as well as skin and bones, are summarized. The disturbances in the ATP amount, its cellular localization, or interaction with target elements may induce pathological changes in signaling pathways leading to the development of serious diseases. The impact of an ATP imbalance on the development of dangerous health dysfunctions such as neurodegeneration diseases, cardiovascular diseases (CVDs), diabetes mellitus, obesity, cancers and immune pathogenesis are discussed here.

Human antioxidant protein 1 (ATOX1) is an essential regulator of copper homeostasis in cells. By interacting with other proteins involved in controlling the intracellular levels of cuprous ions (Cu⁺), ATOX1 contributes to the import, export, and subcellular distribution of Cu⁺ as it functions within the CTR1-ATOX1-ATP7A/ATP7B axis. For this reason, ATOX1 plays a key role in preventing copper toxicity. Since copper ions have been shown to regulate the activity of a subset of other signaling proteins, ATOX1 can support cell proliferation, migration, and survival. Notably, ATOX1 is the only identified copper chaperone that has transcription factor activity. In this respect, CCND1, MDC1, NCF1, PPA2, and SOD3 have been experimentally validated as transcriptional targets of ATOX1 in distinct types of cells. The multifaceted actions of ATOX1 indicate that its dysregulation can lead to changes in the activity of crucial signaling pathways associated with diverse disorders, including cancer. Indeed, ATOX1 levels are frequently increased in cancer as demonstrated in multiple studies and supported by data available in GEPIA. ATOX1 has been implicated in cancer biology because of its role in the proliferation and metastatic spread of cancer cells and protection from oxidative stress. Additionally, ATOX1 may impact the drug response and resistance of cancer cells by influencing detoxification mechanisms as demonstrated for platinum-based therapies. In turn, the role of ATOX1 in the susceptibility of cancer cells to targeted therapies and immunotherapy remains elusive. This, however, should be a direction of further research considering the recent advances in understanding the complex role of copper in cancer cells, which can be associated with either protumorigenic effects (cuproplasia) or the induction of novel copper-dependent regulated cell death (cuproptosis) to combat cancer cells. Therefore, the disruption of ATOX1-mediated processes could be beneficial for the efficacy of anticancer therapies, although this possibility should be treated with caution because of the dual role of copper in cancer. Moreover, the prognostic value of ATOX1 expression for the clinical outcome of cancer patients needs to be clarified. In this review, we summarize the current state of knowledge about ATOX1 in cancer focusing on its molecular aspects and potential clinical implications.

New boron carriers with high boron content and targeted cancer-cell delivery are considered the first choice for boron neutron capture therapy (BNCT) for cancer treatment. Previously, we have shown that composites of antisense oligonucleotide and boron clusters are functional nanoparticles for the downregulation of expression of epidermal growth factor receptor (EGFR) and can be loaded into EGFR-overexpressing cancer cells without a transfection factor. In this study, we hypothesize that free cellular uptake is mediated by binding and activation of the EGFR by boron clusters. Proteomic analysis of proteins pulled-down from various EGFR-overexpressing cancer cells using short oligonucleotide probes, conjugated to 1,2-dicarba-closo-dodecaborane (1,2-DCDDB, [C₂B₁₀H₁₂]) and [(3,3′-Iron-1,2,1′,2′-dicarbollide)⁻] (FESAN, [Fe(C₂B₉H₁₁)₂]⁻), evidenced that boron cage binds to EGFR subdomains. Moreover, inductively coupled plasma mass spectrometry (ICP MS) and fluorescence microscopy analyses confirmed that FESANs-highly decorated B-ASOs were efficiently delivered and internalized by EGFR-overexpressing cells. Antisense reduction of EGFR in A431 and U87-MG cells resulted in decreased boron accumulation compared to control cells, indicating that cellular uptake of B-ASOs is related to EGFR-dependent internalization. The data obtained suggest that EGFR-mediated cellular uptake of B-ASO represents a novel strategy for cellular delivery of therapeutic nucleic acids (and possibly other medicines) conjugated to boron clusters.

Boron cluster-conjugated antisense oligonucleotides (B-ASOs) have already been developed as therapeutic agents with “two faces”, namely as potential antisense inhibitors of gene expression and as boron carriers for boron neutron capture therapy (BNCT). The previously observed high antisense activity of some B-ASOs targeting the epidermal growth factor receptor (EGFR) could not be rationally assigned to the positioning of the boron cluster unit: 1,2-dicarba-closo-dodecaborane (0), [(3,3′-Iron-1,2,1′,2′-dicarbollide) (1-), FESAN], and dodecaborate (2-) in the ASO chain and its structure or charge. For further understanding of this observation, we performed systematic studies on the efficiency of RNase H against a series of B-ASOs models. The results of kinetic analysis showed that pyrimidine-enriched B-ASO oligomers activated RNase H more efficiently than non-modified ASO. The presence of a single FESAN unit at a specific position of the B-ASO increased the kinetics of enzymatic hydrolysis of complementary RNA more than 30-fold compared with unmodified duplex ASO/RNA. Moreover, the rate of RNA hydrolysis enhanced with the increase in the negative charge of the boron cluster in the B-ASO chain. In conclusion, a “smart” strategy using ASOs conjugated with boron clusters is a milestone for the development of more efficient antisense therapeutic nucleic acids as inhibitors of gene expression.

Two, well defined binary crystals with 1-Hydroxy-4,5-Dimethyl-Imidazole 3-Oxide (HIMO) as coformer and thiobarbituric acid (TBA) as well barbituric acid (BA) as Active Pharmaceutical Ingredients (APIs) were obtained by cocrystallization (from methanol) or mechanochemically by grinding. The progress of cocrystal formation in a ball mill was monitored by means of high-resolution, solid state NMR spectroscopy. The ¹³C CP/MAS, ¹⁵N CP/MAS and ¹H Very Fast (VF) MAS NMR procedures were employed to inspect the tautomeric forms of the APIs, structure elucidation of the coformer and the obtained cocrystals. Single crystal X-ray studies allowed us to define the molecular structure and crystal packing for the coformer as well as the TBA/HIMO and BA/HIMO cocrystals. The intermolecular hydrogen bonding, π–π interactions and CH-π contacts responsible for higher order organization of supramolecular structures were determined. Biological studies of HIMO and the obtained cocrystals suggest that these complexes are not cytotoxic and can potentially be considered as therapeutic materials.

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO’s properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.