Search Results (51)

Background and Objectives: Despite the development of treatment methods and the emergence of alternative new approaches in recent years, the visual prognosis of retinoblastoma contains deficiencies and this situation increases the need for the development of new treatment approaches. The cytotoxic and apoptosis-inducing effects of the combination of boswellic acid (BA), which has been determined to have significant potential in preclinical and clinical studies of various diseases, and Cisplatin (Cis), a potent chemotherapy agent, were investigated on the human retinoblastoma cell line (Y79). Materials and Methods: The cytotoxic effect of BA and Cis on Y79 cells was determined by the water soluble tetrazolium-1 (WST-1) test, the apoptotic rate of the cells was determined by annexin V staining, and the gene expressions of Protein53 (p53), Caspase-3 and Nuclear factor kappa B (NF-κB), which play an important role in apoptosis, were determined by RT-qPCR analysis. Interleukin 1-beta (IL1-β), tumor necrosis factor-α (TNF-α) and interferon γ (IFN-γ) levels were analyzed in cell lysates obtained from the experimental groups. Results: The combination of BA and Cis selectively inhibited the growth of Y79 cells and modulated NF-κB signaling, potentially through post-translational regulatory mechanisms. Moreover, it induced apoptosis by increasing p53 and Caspase-3 expressions, confirming its pro-apoptotic effects. Additionally, the combination treatment was associated with a reduction in inflammatory cytokine levels (TNF-α, IL1-β), suggesting a potential regulatory effect on inflammation-related pathways rather than direct inhibition of NF-κB activation. Conclusions: These findings suggest that BA combined with Cis inhibits Y79 retinoblastoma cell growth by inducing apoptosis and modulating NF-κB signaling. While NF-κB mRNA levels increased, reduced inflammatory cytokines and enhanced apoptosis suggest potential post-translational regulation. Further studies are needed to confirm NF-κB protein-level effects and in vivo efficacy. Full article

Intraocular malignant tumors are rare; however, they can cause serious life-threatening complications. Uveal melanoma (UM) and retinoblastoma (RB) are the most common intraocular tumors in adults and children, respectively, and come with a great disease burden. For many years, several different treatment modalities for UM and RB have been proposed, with chemotherapy for RB cases and plaque radiation therapy for localized UM as first-line treatment options. Extraocular extension, recurrence, and metastasis constitute the major challenges of conventional treatments. To overcome these obstacles, immunotherapy, which encompasses different treatment options such as oncolytic viruses, antibody-mediated immune modulations, and targeted immunotherapy, has shown great potential as a novel therapeutic tool for cancer therapy. These anti-cancer treatment options provide numerous advantages such as selective cancer cell death and the promotion of an anti-tumor immune response, and they prove useful in preventing vision impairment due to macular and/or optic disc involvement. Numerous factors such as the vector choice, route of administration, dosing, and patient characteristics must be considered when engineering an oncolytic virus or other forms of immunotherapy vectors. This manuscript provides an in-depth review of the molecular design of oncolytic viruses (e.g., virus capsid proteins and encapsulation technologies, vectors for delivery, cell targeting) and immunotherapy. The most recent advances in preclinical- and clinical-phase studies are further summarized. The recent developments in virus-like drug conjugates (i.e., AU011), oncolytic viruses for metastatic UM, and targeted immunotherapies have shown great results in clinical trials for the future clinical application of these novel technologies in the treatment algorithm of certain intraocular tumors. Full article

Inonotus obliquus (Chaga) is a medicinal mushroom with several pharmacological properties that is used as a tea in traditional Chinese medicine. In this study, Chaga water extract was digested in vitro to mimic the natural processing and absorption of its biocomponents when it is consumed as functional beverage, and its anticancer activities were evaluated in breast cancer (BC) cell lines, representing HER2-positive and triple-negative subtypes. After chemical characterization by liquid chromatography/mass spectrometry (HR-QTOF) analysis, the effect of Chaga biocomponents on cell viability and cell cycle progression was assessed by MTT assay, FACS analysis, and Western blot. Dihydrofolate reductase (DHFR) activity was measured by an enzymatic assay. Four highly bioactive triterpenoids (inotodiol, trametenolic acid, 3-hydroxy-lanosta-8,24-dien-21-al, and betulin) were identified as the main components, able to decrease BC cell viability and block the cell cycle in G0/G1 by inducing the downregulation of cyclin D1, CDK4, cyclin E, and phosphorylated retinoblastoma protein. DHFR was identified as their crucial target. Moreover, bioactive Chaga components exerted a synergistic action with cisplatin and with trastuzumab in SK-BR-3 cells by inhibiting both HER2 and HER1 activation and displayed an immunomodulatory effect. Thus, Inonotus obliquus represents a source of triterpenoids that are effective against aggressive BC subtypes and display properties of targeted drugs. Full article

Recent advancements in aqueous humor (AH) cell-free DNA (cfDNA) genomics have opened new avenues for ex vivo molecular profiling of retinoblastoma (RB), the most common pediatric intraocular malignancy, where biopsy is typically prohibited. While these insights offer a genetic blueprint of the tumor, they lack multi-omic molecular phenotyping, which is essential for understanding the functional state. Extracellular vesicles (EVs), naturally present in AH, are promising by offering time-resolved phenotypic information. We employed multiplex bead-based flow cytometry and Single Extracellular Vesicle Nanoscopy (SEVEN) to analyze EV phenotypes in AH from a cohort of five RB, with three uveal melanoma (UM) and two age-matched glaucoma (GLC) samples serving as controls. The studies identified CD133-enriched EVs uniquely in RB AH, absent in both GLC and UM AH. This was corroborated by further analysis of five RB cell lines, including two commercial (Y79, Weri) and three in-house developed lines, confirming CD133 enrichment and supporting its role as an RB-specific EV marker. Single-vesicle analysis demonstrated a strong association of CD133 with CD81 and CD63, with minimal CD9 presence. These results, validated through complementary techniques, position CD133 as a critical marker in RB-derived EVs, paving the way for enhanced multi-omic RB characterization and potential advancements in clinical diagnostics. Full article

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6. IPA analysis of these DEGs suggested that FABP5 may be involved in essential roles required for cell development, cell survival and cell homeostasis. In support of this possibility, both mitochondrial and glycolytic functions of HNPCE cells, in which mRNA expression of FABP5, but not that of FABP7, was detected, were shown by using a Seahorse XFe96 Bioanalyzer to be dramatically suppressed by FABP ligand 6-induced inhibition of the activity of FABP5. Furthermore, in IPA upstream analysis, various unfolded protein response (UPR)-related factors were identified as upstream and causal network master regulators. Analysis by qPCR analysis showed significant upregulation of the mRNA expression of most of UPR-related factors and aquaporin1 (AQP1). The findings in this study suggest that HNPCE is one of intraocular cells producing FABP5 and may be involved in the maintenance of UPR and AQP1-related functions of HNPCE. Full article

Small cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma accounting for 15% of lung cancers with dismal survival outcomes. Minimal changes in therapy and prognosis have occurred in SCLC for the past four decades. Recent progress in the treatment of extensive-stage disease (ES-SCLC) has been marked by incorporating immune checkpoint inhibitors (ICIs) into platinum-based chemotherapy, leading to modest improvements. Moreover, few second-line-and-beyond treatment options are currently available. The main limitation for the molecular study of SCLC has been the scarcity of samples, because only very early diseases are treated with surgery and biopsies are not performed when the disease progresses. Despite all these difficulties, in recent years we have come to understand that SCLC is not a homogeneous disease. At the molecular level, in addition to the universal loss of retinoblastoma (RB) and TP53 genes, a recent large molecular study has identified other mutations that could serve as targets for therapy development or patient selection. In recent years, there has also been the identification of new genetic subtypes which have shown us how intertumor heterogeneity exists. Moreover, SCLC can also develop intratumoral heterogeneity linked mainly to the concept of cellular plasticity, mostly due to the development of resistance to therapies. The aim of this review is to quickly present the current standard of care of ES-SCLC, to focus on the molecular landscapes and subtypes of SCLC, subsequently present the most promising therapeutic strategies under investigation, and finally recap the future directions of ongoing clinical trials for this aggressive disease which still remains a challenge. Full article

Retinoblastoma (RB) is the most common malignant intraocular tumor in early childhood. Gene expression profiling revealed that the gastric inhibitory polypeptide receptor (GIPR) is upregulated following trefoil factor family peptide 1 (TFF1) overexpression in RB cells. In the study presented, we found this G protein-coupled transmembrane receptor to be co-expressed with TFF1, a new diagnostic and prognostic RB biomarker for advanced subtype 2 RBs. Functional analyses in two RB cell lines revealed a significant reduction in cell viability and growth and a concomitant increase in apoptosis following stable, lentiviral GIPR overexpression, matching the effects seen after TFF1 overexpression. In chicken chorioallantoic membrane (CAM) assays, GIPR-overexpressing RB cells developed significantly smaller CAM tumors. The effect of GIPR overexpression in RB cells was reversed by the GIPR inhibitor MK0893. The administration of recombinant TFF1 did not augment GIPR overexpression effects, suggesting that GIPR does not serve as a TFF1 receptor. Investigations of potential GIPR up- and downstream mediators suggest the involvement of miR-542-5p and p53 in GIPR signaling. Our results indicate a tumor suppressor role of GIPR in RB, suggesting its pathway as a new potential target for future retinoblastoma therapy. Full article

Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the “classical” protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and—dependent on the cell line—AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma. Full article

Cervical cancer is a major cause of death in women despite the advancement of current treatment modalities. The conventional therapeutic agent, cisplatin (CCDP), is the standard treatment for CC; however, resistance often develops due to the cancer’s heterogeneity. Therefore, a detailed elucidation of the specific molecular mechanisms driving CC is crucial for the development of targeted therapeutic strategies. Retinoblastoma binding protein 6 (RBBP6) is a potential biomarker associated with cell proliferation and is upregulated in cervical cancer sites, exhibiting apoptosis and dysregulated p53 expression. Furthermore, RBBP6 has been demonstrated to sensitize cancer cells to radiation and certain chemotherapeutic agents by regulating the Bcl-2 gene, thus suggesting a crosstalk among RBBP6/p53/BCL-2 oncogenic signatures. The present study, therefore, investigated the relationship between cisplatin and RBBP6 expression in CC cells. Herein, we first explored bioinformatics simulations and identified that the RBBP6/p53/BCL-2 signaling pathway is overexpressed and correlated with CC. For further analysis, we explored the Genomics of Drug Sensitivity in Cancer (GDSC) and found that most of the CC cell lines are sensitive to CCDP. To validate these findings, RBBP6 was silenced in HeLa and Vero cells using RNAi technology, followed by measurement of wild-type p53 and Bcl-2 at the mRNA level using qPCR. Cells co-treated with cisplatin and siRBBP6 were subsequently analyzed for apoptosis induction and real-time growth monitoring using flow cytometry and the xCELLigence system, respectively. Cancer cells in the co-treatment group showed a reduction in apoptosis compared to the cisplatin-treated group. Moreover, the real-time growth monitoring revealed a reduced growth rate in RBBP6 knockdown cells treated with cisplatin. Although wild-type p53 remained unchanged in the co-treatment group of cancer cells, Bcl-2 was completely repressed, suggesting that RBBP6 is necessary for sensitizing cervical cancer cells to cisplatin treatment by downregulating Bcl-2. The Vero cell population, which served as a non-cancerous control cell line in this study, remained viable following treatment with both siRBBP6 and cisplatin. Findings from this study suggest that RBBP6 expression promotes cisplatin sensitivity in HeLa cells through Bcl-2 downregulation. Knockdown of RBBP6 limits apoptosis induction and delays cell growth inhibition in response to cisplatin. The knowledge obtained here has the potential to help improve cisplatin efficacy through personalized administration based on the expression profile of RBBP6 among individual patients. Full article

In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca²⁺ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca²⁺ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca²⁺ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca²⁺ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk. Full article

This study explores the impact of antiretroviral administration on the expression of human endogenous retroviruses (HERVs), cell growth, and invasive capability of human melanoma cell lines in culture. We investigated three antiretrovirals—lamivudine, doravirine, and cabotegravir—in A375, FO-1, and SK-Mel-28, BRAF-mutated, and in MeWo, P53-mutated, melanoma cell lines. The findings indicate a general capability of these drugs to downregulate the expression of HERV-K Pol and Env genes and hinder cell viability, mobility, and colony formation capacity of melanoma cells. The antiretroviral drugs also demonstrate selectivity against malignant cells, sparing normal human epithelial melanocytes. The study reveals that the integrase inhibitor cabotegravir is particularly effective in inhibiting cell growth and invasion across different cell lines in comparison with lamivudine and doravirine, which are inhibitors of the viral reverse transcriptase enzyme. The investigation further delves into the molecular mechanisms underlying the observed effects, highlighting the potential induction of ferroptosis, apoptosis, and alterations in cell cycle regulatory proteins. Our findings showed cytostatic effects principally revealed in A375, and SK-Mel-28 cell lines through a downregulation of retinoblastoma protein phosphorylation and/or cyclin D1 expression. Signs of ferroptosis were detected in both A375 cells and FO-1 cells by a decrease in glutathione peroxidase 4 and ferritin expression, as well as by an increase in transferrin protein levels. Apoptosis was also detected in FO-1 and SK-Mel-28, but only with cabotegravir treatment. Moreover, we explored the expression and activity of the stimulator of interferon genes (STING) protein and its correlation with programmed death-ligand 1 (PD-L1) expression. Both the STING activity and PD-L1 expression were decreased, suggesting that the antiretroviral treatments may counteract the detrimental effects of PD-L1 expression activation through the STING/interferon pathway triggered by HERV-K. Finally, this study underscores the potential therapeutic significance of cabotegravir in melanoma treatment. The findings also raise the prospect of using antiretroviral drugs to downregulate PD-L1 expression, potentially enhancing the therapeutic responses of immune checkpoint inhibitors. Full article

Retinoblastoma represents the most prevalent malignant neoplasm affecting the eyes in childhood. The clear-cut origin of retinoblastoma has not yet been determined; however, based on experiments, it has been suggested that RB1 loss in cone photoreceptors causes retinoblastoma. Pituitary adenylate-cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide which has been shown to be affected in certain tumorous transformations, such as breast, lung, kidney, pancreatic, colon, and endocrine cancers. This study aimed to investigate potential changes in both PACAP38 and PAC1 receptor (PAC1R) expression in human retinoblastoma and the effect of PACAP38 administration on the survival of a human retinoblastoma cell line (Y-79). We analyzed human enucleation specimens removed because of retinoblastoma for PACAP38 and PAC1R immunostaining and the effect of PACAP38 on the survival of the Y-79 cell line. We described for the first time that human retinoblastoma cells from patients showed only perinuclear, dot-like immunopositivity for both PACAP38 and PAC1R, irrespective of laterality, genetic background, or histopathological features. Nanomolar (100 nM and 500 nM) PACAP38 concentrations had no effect on the viability of Y-79 cells, while micromolar (2 µM and 6 µM) PACAP38 significantly decreased tumor cell viability. These findings, along with general observations from animal studies showing that PACAP38 has strong anti-apoptotic, anti-inflammatory, and antioxidant effects on ocular tissues, together suggest that PACAP38 and its analogs are promising candidates in retinoblastoma therapy. Full article

This study investigated the effects of progesterone receptors A (PRA) and B (PRB) on proliferation, migration, invasion, anchorage-independent growth (AIG), and apoptosis of FE25 cells, a precancer p53- and retinoblastoma-defective human fallopian tube epithelial cell line. We observed that the transfection of PRA (FE25-PRA) or PRB (FE25-PRB) into FE25 cells significantly increased the expression of PRA or PRB at both RNA and protein levels without affecting cell morphology. The FE25-PRA cells exhibited slower proliferation, whereas FE25-PRB showed faster cell proliferation than the control cells. In contrast, the FE25-PRA cells showed the highest migration and invasion abilities, whereas the FE25-PRB cells showed the lowest migration and invasion abilities. After treatment with progesterone, all cell types showed decreased AIG levels, increased apoptotic rates in Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling assay (TUNEL) staining, and increased levels of apoptotic proteins ascertained based on cleaved caspase-3 levels. The half-maximal inhibitory concentration of carboplatin increased in FE25-PRB cells, but that of paclitaxel remained unchanged. Overall, this study suggests that PRA and PRB have distinct roles in regulating the behavior of FE25 cells, and targeting these receptors could be a potential therapeutic strategy for ovarian cancer treatment. If PRA or PRB overexpression is observed in high-grade serous carcinoma, progesterone could be considered as an adjuvant therapy for these specific cancer patients. However, further research is needed to confirm these findings and investigate the mechanisms underlying these effects. Full article

Hyperforin (HPF) is an acylphloroglucinol compound found abundantly in Hypericum perforatum extract which exhibits antidepressant, anti-inflammatory, antimicrobial, and antitumor activities. Our recent study revealed a potent antimelanoma effect of HPF, which hinders melanoma cell proliferation, motility, colony formation, and induces apoptosis. Furthermore, we have identified glutathione peroxidase-4 (GPX-4), a key enzyme involved in cellular protection against iron-induced lipid peroxidation, as one of the molecular targets of HPF. Thus, in three BRAF-mutated melanoma cell lines, we investigated whether iron unbalance and lipid peroxidation may be a part of the molecular mechanisms underlying the antimelanoma activity of HPF. Initially, we focused on heme oxygenase-1 (HO-1), which catalyzes the heme group into CO, biliverdin, and free iron, and observed that HPF treatment triggered the expression of this inducible enzyme. In order to investigate the mechanism involved in HO-1 induction, we verified that HPF downregulates the BTB and CNC homology 1 (BACH-1) transcription factor, an inhibitor of the heme oxygenase 1 (HMOX-1) gene transcription. Remarkably, we observed a partial recovery of cell viability and an increase in the expression of the phosphorylated and active form of retinoblastoma protein when we suppressed the HMOX-1 gene using HMOX-1 siRNA while HPF was present. This suggests that the HO-1 pathway is involved in the cytostatic effect of HPF in melanoma cells. To explore whether lipid peroxidation is induced, we conducted cytofluorimetric analysis and observed a significant increase in the fluorescence of the BODIPY C-11 probe 48 h after HPF administration in all tested melanoma cell lines. To discover the mechanism by which HPF triggers lipid peroxidation, along with the induction of HO-1, we examined the expression of additional proteins associated with iron homeostasis and lipid peroxidation. After HPF administration, we confirmed the downregulation of GPX-4 and observed low expression levels of SLC7A11, a cystine transporter crucial for the glutathione production, and ferritin, able to sequester free iron. A decreased expression level of these proteins can sensitize cells to lipid peroxidation. On the other hand, HPF treatment resulted in increased expression levels of transferrin, which facilitates iron uptake, and LC3B proteins, a molecular marker of autophagy induction. Indeed, ferritin and GPX-4 have been reported to be digested during autophagy. Altogether, these findings suggest that HPF induced lipid peroxidation likely through iron overloading and decreasing the expression of proteins that protect cells from lipid peroxidation. Finally, we examined the expression levels of proteins associated with melanoma cell invasion and metastatic potential. We observed the decreased expression of CD133, octamer-4, tyrosine-kinase receptor AXL, urokinase plasminogen activator receptor, and metalloproteinase-2 following HPF treatment. These findings provide further support for our previous observations, demonstrating the inhibitory effects of HPF on cell motility and colony formation in soft agar, which are both metastasis-related processes in tumor cells. Full article

Cyclin-dependent kinase 2 (CDK2) has been garnering considerable interest as a target to develop new cancer treatments and to ameliorate resistance to CDK4/6 inhibitors. However, a selective CDK2 inhibitor has yet to be clinically approved. With the desire to discover novel, potent, and selective CDK2 inhibitors, the phenylsulfonamide moiety of our previous lead compound 1 was bioisosterically replaced with pyrazole derivatives, affording a novel series of N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amines that exhibited potent CDK2 inhibitory activity. Among them, 15 was the most potent CDK2 inhibitor (K_i = 0.005 µM) with a degree of selectivity over other CDKs tested. Meanwhile, this compound displayed sub-micromolar antiproliferative activity against a panel of 13 cancer cell lines (GI₅₀ = 0.127–0.560 μM). Mechanistic studies in ovarian cancer cells revealed that 15 reduced the phosphorylation of retinoblastoma at Thr821, arrested cells at the S and G2/M phases, and induced apoptosis. These results accentuate the potential of the N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amine scaffold to be developed into potent and selective CDK2 inhibitors for the treatment of cancer. Full article