The protein disulfide isomerase A3 (PDIA3) is directly or indirectly involved in various physiopathological processes and participates in cancer initiation, progression and chemosensitivity. However, little is known about its involvement in glioblastoma. To obtain specific information, we performed cellular experiments in the T98G and U−87 MG glioblastoma cell lines to evaluate the role of PDIA3. The loss of PDIA3 functions, either through inhibition or silencing, reduced glioblastoma cells spreading by triggering cytotoxic phenomena. PDIA3 inhibition led to a redistribution of PDIA3, resulting in the formation of protein aggregates visualized through immunofluorescence staining. Concurrently, cell cycle progression underwent arrest at the G₁/S checkpoint. After PDIA3 inhibition, ROS-independent DNA damage and the activation of the repair system occurred, as evidenced by the phosphorylation of H2A.X and the overexpression of the Ku70 protein. We also demonstrated through a clonogenic assay that PDIA3 inhibition could increase the chemosensitivity of T98G and U-87 MG cells to the approved glioblastoma drug temozolomide (TMZ). Overall, PDIA3 inhibition induced cytotoxic effects in the analyzed glioblastoma cell lines. Although further in vivo studies are needed, the results suggested PDIA3 as a novel therapeutic target that could also be included in already approved therapies.

PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3.

Human chemical carcinogenesis is a multistage process where chemicals or their metabolites cause irreversible changes in normal cell physiology, eventually leading to uncontrolled proliferation, transforming a normal cell into a cancerous one. Signal transducer and activator of transcription 3 (STAT3) is a cytoplasmic transcription factor that regulates cell proliferation, differentiation, apoptosis, angiogenesis, inflammation, and immune responses. Its aberrant activation triggers tumor progression by promoting the expression of oncogenic genes; thus, STAT3 is classified as an oncoprotein. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that responds to a wide variety of chemicals, including carcinogens like dioxins, inducing genes associated with detoxification, proliferation, and immune regulation. Recent reports show that AhR plays a critical role in cancer development and maintenance. AhR may interact with signaling pathways, like the STAT3 pathway, which mediates the carcinogenic effects of several pollutants. Various chemical agents, such as industrial waste and hydrocarbon compounds, can alter the expression or signaling activity of AhR and STAT3 pathways, leading to different types of cancers. Understanding the complex STAT3-AhR network in the regulation of chemical carcinogenesis could open new avenues for cancer prevention or treatment, particularly in personalized medicine, aiming to improve life expectancy and achieving a complete cure.

Organochlorine pesticides (OCPs) are a class of environmentally persistent and bioaccumulative pollutants. Among these, β-hexachlorocyclohexane (β-HCH) is a byproduct of lindane synthesis, one of the most worldwide widespread pesticides. β-HCH cellular mechanisms inducing chemical carcinogenesis correspond to many of those inducing chemoresistance, in particular, by the activation of signal transducer and activator of transcription 3 (STAT3) signaling pathways. For this purpose, four cell lines, representative of breast, lung, prostate, and hepatocellular cancers, were treated with β-HCH, specific tyrosine kinase inhibitors (TKIs), and a STAT3 inhibitor. All cell samples were analyzed by a viability assay, immunoblotting analysis, a wound-healing assay, and a colony formation assay. The results show that β-HCH reduces the efficacy of TKIs. The STAT3 protein, in this context, plays a central role. In fact, by inhibiting its activity, the efficacy of the anticancer drug is restored. Furthermore, this manuscript aimed to draw the attention of the scientific and socio-healthcare community to the issue of prolonged exposure to contaminants and their impact on drug efficacy.

Hormone-independent breast and prostate cancers represent highly aggressive malignancies characterized by profound metabolic reprogramming, elevated oxidative stress, and loss of sensitivity to endocrine therapies. Increasing evidence indicates that tumor progression and metabolic plasticity are sustained by interconnected signaling networks linking transcriptional regulation to energy metabolism. Among these, the STAT3–PKM2–HIF-1α signaling axis, functionally reinforced by reactive oxygen species (ROS), has been proposed as a central regulator of the Warburg phenotype and cellular adaptation to adverse microenvironmental conditions. Using androgen-independent prostate cancer (DU145) and triple-negative breast cancer (KPL-4) cell lines, we demonstrated constitutive activation and reciprocal regulation of STAT3, PKM2, and HIF-1α. Pharmacological inhibition of STAT3, stabilization of tetrameric PKM2 by L-serine, and ROS scavenging with N-acetylcysteine significantly reduced STAT3 phosphorylation, PKM2 nuclear translocation, and HIF-1α stabilization. These molecular effects were accompanied by decreased intracellular ROS levels, reduced lactate production, increased pyruvate levels, and a metabolic shift toward oxidative phosphorylation. Functionally, treated cells exhibited reduced Ki-67 expression and impaired clonogenic capacity. Our results identify the STAT3–PKM2–HIF-1α/ROS axis as a key determinant of metabolic and phenotypic plasticity in hormone-independent breast and prostate cancers, highlighting its potential as a molecular target for therapeutic modulation of cancer-associated metabolic phenotypes.