Achilleas Mitrakas

Background/Objectives: The enhancement of antitumor immune responses by radiotherapy (RT) is partially attributed to the activation of the IFN-type-I pathway. However, the loss of HLA-class-I molecules, which occurs in a large percentage of non-small-cell lung cancers (NSCLCs), may block the cytotoxic effect of T-cells and immunotherapy (IO). Moreover, autophagy is also involved in HLA downregulation. We investigated the complex interactions between RT, HLA molecules, autophagy, and IFN-type-I responses. Methods: The A549, H1299, and ATG7-deficient NSCLC cell lines, along with the modified shLC3A H1299 cell line, were used for in vitro experiments. The effect of RT (8 and 3 × 8 Gy) on Interferon beta (IFNβ), IFN-stimulated genes (ISGs), and HLA-class-I expression in combination with IFN-type-I-response inhibitors (Ruxolitinib, Tofacitinib, Amlexanox) targeting the JAK and TBK1 was studied with Flow cytometry and RT-PCR. Results: RT significantly induced HLA-class-I expression. A parallel upregulation of IFNβ and ISGs mRNA levels was also documented. Although the IFN-type-I-response inhibitors suppressed the RT-induced IFNβ and ISGs expression, their effect on HLA-class-I expression was minimal. Blockage of LC3A autophagy (shLC3A cell line) significantly upregulated HLA-class-I basal levels, and RT further enhanced HLA expression. IFN-type-I-response inhibitors blocked the RT-inductive effect in the shLC3A H1299, but had no effect in the ATG7-deficient H1650 cell line. Conclusions: The current study supports the theory that baseline autophagy, RT-induced autophagy blockage, and IFN-type-I response enhancement define the HLA-class-I levels in NSCLC cells. This complex interplay emerges as a promising target for the development of radio-vaccination strategies to enhance the efficacy of radio-immunotherapy.

Male infertility affects nearly 15% of couples worldwide, with chromosomal abnormalities representing a major underlying cause. This review explores how numerical and structural chromosomal anomalies, along with environmental exposures, lifestyle factors, and age-related genetic changes, disrupt spermatogenesis and contribute to infertility. It synthesizes findings from cytogenetic, molecular, and clinical studies, with particular focus on mechanisms such as meiotic nondisjunction, spindle assembly checkpoint dysfunction, and alterations in cohesin and synaptonemal complex proteins. Chromosomal abnormalities, both numerical and structural, emerge as key contributors to male infertility by impairing chromosomal segregation and recombination, often leading to azoospermia or oligospermia. Meiotic checkpoint failures and recombination errors further exacerbate the production of aneuploid sperm. Environmental toxins, oxidative stress, and poor nutrition disrupt hormonal balance and chromatin integrity, while advancing paternal age is associated with increased sperm aneuploidy and impaired meiotic control, with implications for assisted reproduction. Specific syndromes, including AZF deletions, Kallmann syndrome, and 46,XX testicular DSD, exemplify the direct genetic impact on male fertility. Overall, chromosomal abnormalities are central to the pathophysiology of male infertility, arising from intrinsic meiotic errors as well as extrinsic environmental and lifestyle factors. Integrating cytogenetic diagnostics, genetic counseling, and lifestyle interventions is essential for comprehensive fertility assessment and management. Further research into molecular biomarkers and targeted therapies could enhance diagnosis, improve treatment strategies, and lead to better reproductive outcomes.

Epilepsy manifests as recurrent spontaneous seizures associated with irregular brain activity. Recognizing the limitations of conventional antiepileptic treatments, we explored the therapeutic potential of essential oils (EOs) derived from Greek aromatic plants (Mentha pulegium, Mentha spicata wild, Mentha piperita, Lavandula angustifolia and Origanum Dictamnus). Specifically, we explored their radical scavenging capacity (DPPH), as well as their antioxidant (AOP and MDA levels) and neuroprotective effect in a PTZ-induced epilepsy Balb/c mice model (animals were pretreated with EOs prior to PTZ treatment). Our results indicated that Mentha piperita emerges as the most promising EO, demonstrating strong antioxidant activity and the highest radical scavenging ability (IC₅₀ = 1.9 mg/mL). Mentha pulegium also exhibited considerable antioxidant potential, demonstrating the strongest effect in the AOP assay when administered prior to PTZ treatment. Furthermore, Origanum dictamnus exhibited the strongest potential to attenuate MDA formation in the presence of PTZ. Finally, immunohistochemistry indicated a trend of neuronal preservation in animals pretreated with EOs prior to PTZ, with Mentha piperita demonstrating the most significant effect. Based on these findings, we suggest that certain EOs possess significant antioxidant and neuroprotective properties. Further research is warranted to validate these results and elucidate the active ingredients responsible for the observed properties.

Background/Objectives: Non-small cell lung cancer (NSCLC) is a major cause of cancer-related deaths globally. The study focuses on understanding the interplay between genetic mutations, cancer stem cells (CSCs), and the tumor microenvironment (TME) in driving NSCLC progression, resistance to therapies, and relapse. Methods: A systematic search was conducted in PubMed and Scopus databases to identify significant and valuable studies relevant to NSCLC, focusing on genetic mutations, CSCs, and the TME. Articles were selected based on their relevance, methodological severity, date of publication, and scientific soundness related to NSCLC biology and therapeutic strategies. This review synthesized findings from these sources to highlight key mechanisms and potential therapeutic interventions. Results: Mutations in critical genes in KRAS, EGFR, TP53, and other key genes interfere with stem cell regulation, promoting CSC-like behavior, resistance to therapy, and immune evasion. The tumor microenvironment (TME), including immune cells, fibroblasts, and extracellular matrix components, further supports tumor growth and reduction in treatment efficacy. Promising strategies, including CSC targeting, TME modulation, and the development of novel biomarkers, have shown potential in preclinical and clinical studies. Conclusions: The association between genetic alterations, CSCs, the TME, and other cellular pathways—including cell metabolism and immune evasion—plays a crucial role in therapy resistance, highlighting the need for comprehensive treatment strategies. The combination of genomic profiling with TME-targeting therapies could lead to personalized treatment approaches, offering hope for better clinical outcomes and reduced mortality in NSCLC patients.

Background/Objectives: Immune checkpoint inhibitors have an established role in non-small cell lung cancer (NSCLC) therapy. The loss of HLA-class-I expression allows cancer cell evasion from immune surveillance, disease progression, and failure of immunotherapy. The restoration of HLA-class-I expression may prove to be a game-changer in current immunotherapy strategies. Autophagic activity has been recently postulated to repress HLA-class-I expression in cancer cells. Methods: NSCLC cell lines (A549 and H1299) underwent late-stage (chloroquine and bafilomycin) and early-stage autophagy blockage (ULK1 inhibitors and MAP1LC3A silencing). The HLA-class-I expression was assessed with flow cytometry, a Western blot, and RT-PCR. NSCLC tissues were examined for MAP1LC3A and HLA-class-I expression using double immunohistochemistry. CD8+ T-cell cytotoxicity was examined in cancer cells pre-incubated with chloroquine and anti-PD-L1 monoclonal antibodies (Moabs); Results: A striking increase in HLA-class-I expression following incubation with chloroquine, bafilomycin, and IFNγ was noted in A549 and H1299 cancer cells, respectively. This effect was further confirmed in CD133+ cancer stem cells. HLA-class-I, β2-microglobulin, and TAP1 mRNA levels remained stable. Prolonged exposure to chloroquine further enhanced HLA-class-I expression. Similar results were noted following exposure to a ULK1 and a PIKfyve inhibitor. Permanent silencing of the MAP1LC3A gene resulted in enhanced HLA-class-I expression. In immunohistochemistry experiments, double LC3A+/HLA-class-I expression was seldom. Pre-incubation of H1299 cancer cells with chloroquine and anti-PD-L1 MoAbs increased the mean % of apoptotic/necrotic cells from 2.5% to 18.4%; Conclusions: Autophagy blockers acting either at late or early stages of the autophagic process may restore HLA-class-I-mediated antigen presentation, eventually leading to enhanced immunotherapy efficacy.

Hepatocellular carcinoma (HCC) is one the most common primary malignancies with high mortality and morbidity. The melanoma-associated antigen (MAGE) gene family includes several genes that are highly expressed in numerous human cancers, making many of them part of the cancer-testis antigen (CTA) family. MAGE-C1 is expressed in various malignancies but is absent in normal cells, except for the male germ line. Its presence is associated with a worse prognosis, increased tumor aggressiveness, and lymph node invasion. Similarly, MAGE-C2 is linked to the development of various malignant tumors. Despite these associations, the roles and mechanisms of MAGE-C1/MAGE-C2 in HCC remain unclear. This study aimed to evaluate the expression of MAGE-C1 and MAGE-C2 in HCC and correlate it with clinicohistological characteristics. Our findings indicated that MAGE-C1 expression is associated with a higher number of nodules, elevated AFP levels, HBV or HCV positivity, older age, male sex, and lymph node invasion. MAGE-C2 expression was correlated with these characteristics and the presence of cirrhosis. These results align with the limited literature, which suggests a correlation between MAGE expression and older age and HBV infection. Consequently, our study suggests that MAGE-C1 and MAGE-C2 are promising novel biomarkers for prognosis and potential therapeutic targets in HCC.

Diacylhydrazine bridged anthranilic acids with aryl and heteroaryl domains have been synthesized as the open flexible scaffold of arylamide quinazolinones in order to investigate flexibility versus rigidity towards DNA photocleavage and sensitivity. Most of the compounds have been synthesized via the in situ formation of their anthraniloyl chloride and subsequent reaction with the desired hydrazide and were obtained as precipitates, in moderate yields. All compounds showed high UV-A light absorption and are eligible for DNA photocleavage studies under this “harmless” irradiation. Despite their reduced UV-B light absorption, a first screening indicated the necessity of a halogen at the p-position in relation to the amine group and the lack of an electron-withdrawing group on the aryl group. These characteristics, in general, remained under UV-A light, rendering these compounds as a novel class of UV-A-triggered DNA photocleavers. The best photocleaver, the compound 9, was active at concentrations as low as 2 μΜ. The 5-Nitro-anthranilic derivatives were inactive, giving the opposite results to their related rigid quinazolinones. Molecular docking studies with DNA showed possible interaction sites, whereas cytotoxicity experiments indicated the iodo derivative 17 as a potent cytotoxic agent and the compound 9 as a slight phototoxic compound.

Biomedical research requires both in vitro and in vivo studies in order to explore disease processes or drug interactions. Foundational investigations have been performed at the cellular level using two-dimensional cultures as the gold-standard method since the early 20th century. However, three-dimensional (3D) cultures have emerged as a new tool for tissue modeling over the last few years, bridging the gap between in vitro and animal model studies. Cancer has been a worldwide challenge for the biomedical community due to its high morbidity and mortality rates. Various methods have been developed to produce multicellular tumor spheroids (MCTSs), including scaffold-free and scaffold-based structures, which usually depend on the demands of the cells used and the related biological question. MCTSs are increasingly utilized in studies involving cancer cell metabolism and cell cycle defects. These studies produce massive amounts of data, which demand elaborate and complex tools for thorough analysis. In this review, we discuss the advantages and disadvantages of several up-to-date methods used to construct MCTSs. In addition, we also present advanced methods for analyzing MCTS features. As MCTSs more closely mimic the in vivo tumor environment, compared to 2D monolayers, they can evolve to be an appealing model for in vitro tumor biology studies.

A set of arylazo sulfones, known to undergo N–S bond cleavage upon light exposure, has been synthesized, and their activity in the dark and upon irradiation towards DNA has been investigated. Their interaction with calf-thymus DNA has been examined, and the significant affinity observed (most probably due to DNA intercalation) was analyzed by means of molecular docking “in silico” calculations that pointed out polar contacts, mainly via the sulfonyl moiety. Incubation with plasmid pBluescript KS II revealed DNA cleavage that has been studied over time and concentration. UV-A irradiation considerably improved DNA damage for most of the compounds, whereas under visible light the effect was slightly lower. Moving to in vitro experiments, irradiation was found to slightly enhance the death of the cells in the majority of the compounds. Naphthylazosulfone 1 showed photo-disruptive effect under UV-A irradiation (IC₅₀ ~13 μΜ) followed by derivatives 14 and 17 (IC₅₀ ~100 μΜ). Those compounds were irradiated in the presence of two non-cancer cell lines and were found equally toxic only upon irradiation and not in the dark. The temporal and spatial control of light, therefore, might provide a chance for these novel scaffolds to be useful for the development of phototoxic pharmaceuticals.

Background: Cancer cells escape macrophage phagocytosis by expressing the CD47 integrin-associated protein that binds to the SIRPα ligand (signal regulatory protein alpha) expressed by macrophages. Immunotherapy targeting this pathway is under clinical development. Methods: We investigated the expression of CD47/SIRPα molecules in a series of 98 NSCLCs, in parallel with the infiltration of tumor stroma by CD68+ macrophages, tumor-infiltrating lymphocytes (TILs), and PD-L1/PD-1 molecules. Results: Extensive membranous CD47 expression by cancer cells characterized 29/98 cases. SIRPα and CD68 were expressed, to a varying extent, by tumor-associated macrophages (Μφ, TAMs). A high CD68Mφ-score in inner tumor areas was linked with improved overall survival (p = 0.005); and this was independent of the stage (p = 0.02, hazard ratio 0.4). In contrast, high SIRPα expression by CD68+ TAMs (SIRPα/CD68-ratio) was linked with CD47 expression by cancer cells, low TIL-score, and poor prognosis (p = 0.02). A direct association of CD47 expression by cancer cells and the % FOXP3+ TILs (p = 0.01, r = 0.25) was also noted. Conclusions: TAMs play an important role in the prognosis of operable NSCLC. As SIRPα+ macrophages adversely affect prognosis, it is suggested that the CD47/SIRPα axis is a sound target for adjuvant immunotherapy policies, aiming to improve the cure rates in operable NSCLC.