P53 Protein Research Articles

Prognostic Importance of PTEN, p53, and MDM2 Expressions in Endometrioid and Serous-Type Endometrial Carcinomas

Aim: Endometrial carcinomas (ECs) are neoplasms with the highest rate of change in the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway. In this study, the relationship among PTEN, MDM2, and p53 protein expression in the PI3K/AKT/mTOR pathway with clinicopathological data in endometrioid endometrial carcinomas (EECs) and serous-type endometrial carcinomas (SECs) was evaluated. Material and Method: A hundred and twenty cases of patients who underwent hysterectomy for EC between 2009 and 2021 were included in the study. Thirty cases of SEC and 90 cases of EEC were evaluated. EEC cases consist of grades 1-3 tumors, and each group includes 30 patients. p53 was examined in two groups as normal/wild type and abnormal/mutant type. PTEN and MDM2 were examined in two groups: positive and negative. The relationship among p53, PTEN, and MDM2 immunohistochemical expression status with histological grade, myometrial invasion, cervical invasion, lymphovascular invasion (LVI), metastatic lymph nodes, presence of tumor in peritoneal fluid, tumor stage, and overall and progression-free survival was evaluated. Results: Loss of PTEN was associated with EEC compared to SEC (p

Genotype–phenotype correlation of SQSTM1 variants in patients with amyotrophic lateral sclerosis

BackgroundSeveral variants of sequestosome 1 (SQSTM1) were screened in patients with amyotrophic lateral sclerosis (ALS), while the pathogenicity and genotype–phenotype correlation remains unclear.MethodsWe screened variants of SQSTM1 gene in 2011...

Clinical Utility of Circulating Tumor DNA for Detecting Lung Cancer Mutations by Targeted Next-Generation Sequencing With Insufficient Tumor Samples.

Circulating tumor deoxyribonucleic acid (ctDNA) is increasingly applied in clinical practice. This study aimed to explore clinical utility of a minimal invasive and sensitive way of ctDNA for next-generation sequencing in non-small cell lung cancer (NSCLC) with inadequate tumor samples. Targeted DNA sequencing was performed on tissue biopsies and matched plasma samples from 60 patients with NSCLC. A total of 13 driving genes were detected in 60 matched tissue DNA (tDNA) and ctDNA samples. Overall concordance rate was 75.47%, with 77.55% sensitivity and 50% specificity. Epidermal growth factor receptor (EGFR) mutations were the most common in both tDNA and ctDNA samples. Among other mutated genes were tumor protein p53 (TP53), erb-b2 receptor tyrosine kinase 2 (ERBB2), anaplastic lymphoma kinase (ALK), cyclin-dependent kinase inhibitor 2A (CDKN2A), ros proto-oncogene 1, and receptor tyrosine kinase (ROS1). Mutations in b-raf proto-oncogene, serine/threonine kinase (BRAF), cluster of differentiation 274 (CD274), neurotrophin receptor tyrosine kinase 1 (NTRK1), and rearranged during transfection (RET) occurred only in plasma. The majority of mutations in both samples were single-nucleotide variants. Deletions were found in EGFR, BRAF, and TP53 in ctDNA, whereas in tDNA, deletions were only found in EGFR. In ALK, single nucleic acid-site amplification occurred simultaneously in tissue and plasma, but insertions and copy number variations were detected only in plasma. Identifying ctDNA mutations by targeted sequencing in plasma is feasible, showing the clinical value of ctDNA-targeted sequencing in NSCLC patients when tumor tissue sampling is insufficient or even impossible.

Peroxyredoxin 6 Protects RIN-M5F Pancreatic Beta Cells Against Streptozotocin-Induced Senescence.

There are evidences that a decrease in the functional activity of pancreatic β-cells under type 2 diabetes conditions may be associated with their senescence, therefore, senotherapy may be a prospective strategy for the diabetes treatment. The senotherapeutic potential of peroxiredoxin 6 (PRDX6) was studied in RIN-m5F pancreatic β-cells with streptozotocin-induced senescence by measuring markers, associated with senescence. Exposure to streptozotocin (STZ) resulted in the senescence of the β-cells. The addition of PRDX6 to the culture medium of RIN-m5F β-cells before treatment with STZ decreased the levels of the following senescence markers: the percentage of SA-β-Gal-positive cells, the phosphorylation of histone H2AX and p21 proteins, and the secretion of the proinflammatory cytokine IL-6 but not the anti-inflammatory cytokine IL-10. These effects were accompanied by a decrease in the production of reactive oxygen species (ROS) and the restoration of impaired NF-κB activation. In addition, PRDX6 altered the production of the heat shock protein HSP90: the production of the constitutive form of HSP90-beta decreased, while the level of inducible HSP90-alpha increased. PRDX6 prevented the senescence of RIN-m5F cells in response to the DNA damage-inducing agent streptozotocin, indicating a potential protective role of PRDX6 in type 2 diabetes mellitus.

Expression of p16 and Ki67 in laryngeal squamous cell carcinoma and their clinical significance.

The objective of this study is to assess the prognostic value of Ki67 and p16 proteins in laryngeal cancer. This retrospective cohort analysis comprised 260 patients diagnosed with laryngeal cancer. Immunohistochemistry (IHC) was employed to assess the expression levels of p16 and Ki67, and their correlation with the survival time of laryngeal cancer patients was analyzed. The Ki67 index level exhibited a significant association with the prognosis of laryngeal cancer. Patients with higher Ki67 index levels demonstrated shorter survival times, more severe pathological classification, and higher tumor stages (P < 0.05). Conversely, no significant differences in prognostic characteristics of laryngeal cancer were observed in the p16 (-/+) population (P > 0.05). The median survival times for patients with Ki67 index levels of 0-35%, 36-70%, and 70-100% were 3.54 years, 2.10 years, and 1.92 years, respectively. After adjusting for age, smoking, alcohol consumption, pathological classification, surgical intervention, recurrence, and metastasis, the risk of death for patients with Ki67 index levels of 70-100% was 2.0504 times higher than that of patients with Ki67 index levels of 0-35% (95% CI: 1.2997-3.2345, P = 0.0020). The Ki67 index level is strongly associated with survival time and the risk of death in laryngeal cancer, making it a valuable prognostic indicator. However, the prognostic value of p16 levels in laryngeal cancer is limited. These findings provide important insights for prognosis evaluation and treatment decision-making in patients with laryngeal cancer.

A mathematical treatment of the impact of p53 signalling on the expression of the transcription factor E2F in the CycE/Cdk2 subsystem in cancerous cells

Complex networks of proteins and genes control cell growth until mitosis. These components play crucial roles in determining the characteristics and timing of each reaction in the cell. A family of cyclins and cyclin-dependent kinases regulates the progression of the cell from one stage to the next, while certain inhibitors regulate cell proliferation, significantly impacting this process. We examine the impact of p53 protein production in the cyclin E/cyclin-dependent kinase 2 (CycE/CDK2) subsystem. Although the precise functioning of the p53 protein is not fully understood, it is known to exhibit pulsatile behaviour, triggered in response to DNA damage. Consequently, the expression of the p53 protein within the cell system increases with the severity of DNA damage. Similarly, levels of CycE/CDK2 are substantially elevated in instances of DNA damage.To investigate this subsystem, we have developed a mathematical model of the CycE/CDK2 subsystem, presented as non-linear ordinary differential equations. Our primary aim is to determine the conditions that could theoretically result in tumorigenesis. We conduct a dynamical systems analysis to investigate the presence of stable limit cycles, which indicate the standard operational states of cells. Our investigation reveals the levels of p53 protein at which the cell can correct defects, undergo apoptosis, or enter quiescence, and where the system exhibits a Hopf-bifurcation (limit cycles). This is the novelty of our study, as previous research has primarily focused on numerical simulations.Using MATLAB simulations, we produced various results in response to different expressions and concentrations of the p53 protein, allowing us to assess its influence on malignant cells. By altering the concentration of protein p53, we ascertain the specific cellular circumstances that facilitate its natural growth, self-correction in the presence of defective activity, and entry into a state of quiescence.This investigation is motivated by the significant role of oscillations in driving, controlling, and managing the cellular oversight machinery. These aspects are incorporated into developing a mathematical model that emulates cellular operations. From this research, we establish that E2F is a critical target to mitigate oncogenic activity, achievable by managing the pulsatile behaviour of the inhibitor p53 or inhibiting its production process within the cell machinery.

Periodic acid-schiff stain and p53 marker: reducing interobserver variability in microinvasive oral squamous cell carcinoma diagnosis.

To assess the effectiveness of periodic acid-Schiff stain and p53 immunohistochemical marker in reducing interobserver variability for diagnosing microinvasive oral squamous cell carcinoma cases. The cross-sectional study was conducted at a tertiary care diagnostic hospital in Rawalpindi, Pakistan, from March 31 to July 31, 2023, and comprised diagnostically challenging biopsy specimens. The specimens were subjected first to haematoxylin and eosin stain, and then with periodic acid-Schiff stain and tumour protein p53 immunohistochemistry simultaneously. A preliminary diagnosis on routine staining alone and a final diagnosis with the two adjuncts were reported by two observers who were both blinded to the prior diagnosis. Data was analysed using SPSS 25. Of the 30 specimens diagnosed, 21 (70%) belonged to males and 9 (30%) to females. The mean age of the patients was 60.47±11.78 years. Periodic acid-Schiff staining and tumour protein p53 immunohistochemistry demonstrated a significant decrease in interobserver variability in the diagnosis of microinvasive oral squamous cell carcinoma, exhibiting enhanced visualisation in basement membrane breach and identifying the invading cells within the lamina propria that were masked on routine staining (p<0.05). Periodic acid-Schiff stain and tumour protein p53 immunohistochemistry could assist in reducing interobserver variability in the diagnosis of microinvasive oral squamous cell carcinoma.

MAPK signaling pathway enhances tolerance of Mytilus galloprovincialis to co-exposure of sulfamethoxazole and polyethylene microplastics

Microplastics (MPs) and antibiotics often coexist in complex marine environments, yet their combined detrimental effects on marine organisms remain underexplored. This study evaluated the effects of polyethylene microplastics (PE, 200 μg/L) and sulfamethoxazole (SMX, 50 μg/L), both individually and in combination, on Mytilus galloprovincialis. The exposure lasted 6 days, followed by a 6-day recovery period. Bioaccumulation, DNA damage, pollutants transport/metabolism related responses and responding alterations of mitogen-activated protein kinase (MAPK) signaling pathway were detected in gills and digestive glands. Bioaccumulation of SMX/PE in mussels occurred in a tissue-specific manner, co-exposure altered SMX contents in investigated tissues. Co-exposure did not induce extra DNA damage, elevated DNA damage was alleviated during the recovery period in all treated groups. The exposure of SMX/PE exerted different alterations in pollutants transport/metabolism related responses, characterized by multixenobiotic resistance and relative expression of key genes (cytochrome P450 monooxygenase, glutathione S-transferase, ATP-binding cassette transporters). Key molecules (p38 MAPK, c-jun N-terminal kinase, extracellular regulated protein kinase, nuclear factor-κB and tumor protein p53) in MAPK signaling pathway were activated at transcriptional and translational levels after SMX/PE and co-exposure. Co-regulation between MAPK members and pollutants transport/metabolism related factors was revealed, suggesting MAPK signaling pathway served as a regulating hub in exposed mussels to conquer SMX/PE stress. Overall, this study provides new insights on SMX/PE induced health risks in marine mussels and potential mechanism through MAPK cascades regulation.

Study on the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells and the intervention effect of N-acetylcysteine

Objective: To investigate the molecular mechanism of autophagy and apoptosis induced by ultrafine carbon black in human bronchial epithelial cells (BEAS-2B cells), and to study the intervention effect and mechanism of N-acetylcysteine (NAC) on ultrafine carbon black-induced oxidative damage in BEAS-2B cells. Methods: In March 2023, BEAS-2B cells were used as research object, an in vitro airway model exposed to ultrafine carbon black was constructed. A control group and three carbon black exposure groups (50, 100, 200 μg/ml) were set up, and the cells were treated with corresponding concentrations of ultrafine carbon black for 24 hours. In addition, the experiment was divided into control group, NAC+ control group, 100 μg/ml carbon black exposure group and NAC+ exposure group. The corresponding groups were treated with 2 mmol/L NAC for 1 h and 100 μg/ml ultrafine carbon black for 24 h, respectively. Cell viability was measured by CCK-8 assay. Intracellular reactive oxygen species (ROS) level was detected by chemical fluorescence method. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), as well as the content of malondialdehyde (MDA) were detected by colorimetry. The mRNA and protein expressions of autophagy-related genes[Atg5, Atg7, Beclin1, microtubule-associated protein light chain 3B (LC3B), p62 and lysosome-associated membrane protein 2 (LAMP2) ] and apoptosis-related genes [B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), Caspase3, Caspase9 and poly (ADP-ribose) polymerase 1 (PARP1) ] were determined by fluorescence quantitative PCR and Western blot. Cell apoptosis was determined by flow cytometry. Results: Compared with the control group, the relative survival rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly decreased, the levels of ROS and MDA were significantly increased, and the activities of SOD, GSH-Px and CAT were significantly decreased (P<0.05). The relative survival rate, ROS and MDA levels, SOD, GSH-Px and CAT activities were significantly correlated with the exposure dose of ultrafine carbon black (r(s)=-0.755, 0.826, 0.934, -0.810, -0.880, -0.840, P<0.05). Compared with the control group, the relative expression levels of Atg5, Atg7, Beclin1, LC3B, p62, LAMP2, Bax, Caspase3, Caspase9, PARP1 mRNA and Atg5, Atg7, Beclin1, LC3BⅡ, p62, LAMP2, Bax, cleaved Caspase3 (C-Caspase3), cleaved Caspase9 (C-Caspase9), cleaved PARP1 (C-PARP1) protein and the ratio of LC3BⅡ/LC3BⅠ in 50, 100 and 200 μg/ml carbon black exposure groups were significantly increased, while the relative expression levels of Bcl-2 mRNA and protein were significantly decreased (P<0.05). The changes of the above indexes were significantly correlated with the exposure dose of carbon black (r(s)=0.892, 0.879, 0.944, 0.892, 0.828, 0.880, 0.814, 0.794, 0.931, 0.918, 0.813, 0.866, 0.774, 0.695, 0.918, 0.761, 0.794, 0.944, 0.833, 0.866, 0.905, -0.886, -0.748, P<0.05). Compared with 100 μg/ml carbon black exposure group, the relative survival rate, the activities of SOD, GSH-Px and CAT in NAC+exposure group were significantly increased, while the levels of ROS and MDA were significantly decreased, and the relative expression levels of LC3B, p62 and Caspase3 mRNA and protein as well as the ratio of LC3BⅡ/LC3BⅠ were significantly decreased, and the differences were statistically significant (P<0.05). Compared with the control group, the apoptosis rates of BEAS-2B cells in 50, 100, 200 μg/ml carbon black exposure groups were significantly increased (P<0.05), and there was a significant positive correlation between ultrafine carbon black exposure dose and cell apoptosis rate (r(s)=0.944, P<0.05). While compared with 100 μg/ml carbon black exposure group, the apoptosis rate of NAC+exposure group was significantly decreased, and the difference was statistically significant (P<0.05) . Conclusion: Cell autophagy and apoptosis may be important pathophysiological mechanisms of ultrafine carbon black-induced oxidative damage in BEAS-2B cells. NAC can alleviate the occurrence of BEAS-2B cell damage caused by ultrafine carbon black by regulating oxidative stress and the cascading autophagy and apoptosis pathways.

Oleanolic acid improves 5-fluorouracil-induced intestinal damage and inflammation by alleviating intestinal senescence

5-Fluorouracil (5-FU) is used as a standard first-line drug for colorectal cancer malignancy (CRC), but it brings a series of side effects such as severe diarrhea and intestinal damage. Our previous study found that a large number of senescent cells increased while 5-Fu induced intestinal damage, and anti-senescence drugs can alleviate its side effects of inflammatory damage. Oleanolic acid (OA) is a common pentacyclic triterpenoid mainly derived from food fungi and medicinal plants, and studies have shown that it mainly possesses hepatoprotective, enzyme-lowering, anti-inflammatory, and anti-tumor effects. But its role in senescence is still unclear. In the present study, we demonstrated for the first time that OA ameliorated 5-Fu-induced human umbilical vein endothelial cells (HUVECs) and human normal intestinal epithelial cells (NCM460) in a 5-Fu-induced cellular senescence model by decreasing the activity of SA-β-gal-positive cells, and the expression of senescence-associated proteins (p16), senescence-associated genes (p53 and p21), and senescence-associated secretory phenotypes (SASPs: IL-1β, IL-6, IL-8, IFN-γ and TNF-α). Meanwhile, in this study, in a BALB/c mouse model, we demonstrated that 5-FU induced intestinal inflammatory response and injury, which was also found to be closely related to the increase of senescent cells, and that OA treatment was effective in ameliorating these adverse phenomena. Furthermore, our in vivo and in vitro studies showed that OA could alleviate senescence by inhibiting mTOR. In colon cancer cell models, OA also enhanced the ability of 5-FU to kill HCT116 cells and SW480 cells. Overall, this study demonstrates for the first time the potential role of OA in counteracting the side effects of 5-FU chemotherapy, providing a new option for the treatment of colorectal cancer to progressively achieve the goal of high efficacy and low toxicity of chemotherapy.

Mutant mice lacking alternatively spliced p53 isoforms unveil Ackr4 as a male-specific prognostic factor in Myc-driven B-cell lymphomas

The Trp53 gene encodes several isoforms of elusive biological significance. Here, we show that mice lacking the Trp53 alternatively spliced (AS) exon, thereby expressing the canonical p53 protein but not isoforms with the AS C-terminus, have unexpectedly lost a male-specific protection against Myc-induced B-cell lymphomas. Lymphomagenesis was delayed in Trp53+/+Eμ-Myc males compared to Trp53ΔAS/ΔAS Eμ-Myc males, but also compared to Trp53+/+Eμ-Myc and Trp53ΔAS/ΔAS Eμ-Myc females. Pre-tumoral splenic cells from Trp53+/+Eμ-Myc males exhibited a higher expression of Ackr4, encoding an atypical chemokine receptor with tumor suppressive effects. We identified Ackr4 as a p53 target gene whose p53-mediated transactivation is inhibited by estrogens, and as a male-specific factor of good prognosis relevant for murine Eμ-Myc-induced and human Burkitt lymphomas. Furthermore, the knockout of ACKR4 increased the chemokine-guided migration of Burkitt lymphoma cells. These data demonstrate the functional relevance of alternatively spliced p53 isoforms and reveal sex disparities in Myc-driven lymphomagenesis.

Mutant mice lacking alternatively spliced p53 isoforms unveil Ackr4 as a male-specific prognostic factor in Myc-driven B-cell lymphomas.

The Trp53 gene encodes several isoforms of elusive biological significance. Here, we show that mice lacking the Trp53 alternatively spliced (AS) exon, thereby expressing the canonical p53 protein but not isoforms with the AS C-terminus, have unexpectedly lost a male-specific protection against Myc-induced B-cell lymphomas. Lymphomagenesis was delayed in Trp53+/+Eμ-Myc males compared to Trp53ΔAS/ΔAS Eμ-Myc males, but also compared to Trp53+/+Eμ-Myc and Trp53ΔAS/ΔAS Eμ-Myc females. Pre-tumoral splenic cells from Trp53+/+Eμ-Myc males exhibited a higher expression of Ackr4, encoding an atypical chemokine receptor with tumor suppressive effects. We identified Ackr4 as a p53 target gene whose p53-mediated transactivation is inhibited by estrogens, and as a male-specific factor of good prognosis relevant for murine Eμ-Myc-induced and human Burkitt lymphomas. Furthermore, the knockout of ACKR4 increased the chemokine-guided migration of Burkitt lymphoma cells. These data demonstrate the functional relevance of alternatively spliced p53 isoforms and reveal sex disparities in Myc-driven lymphomagenesis.

Mutant p53 Misfolding and Aggregation Precedes Transformation into High-Grade Serous Ovarian Carcinoma.

High Grade Serous Ovarian Cancer (HG-SOC), the most prevalent and aggressive gynecological malignancy, is marked by ubiquitous loss of functional p53, largely due to point mutations that arise very early in carcinogenesis. These mutations often lead to p53 protein misfolding and subsequent aggregation, yet the alterations in intracellular p53 dynamics throughout ovarian cancer progression remain poorly understood. HG-SOC originates from the fallopian tube epithelium, with a well-documented stepwise progression beginning with early pre-malignant p53 signatures. These signatures represent largely normal cells that express and accumulate mutant p53, which then transform into benign serous tubal intraepithelial lesions (STIL), progress into late pre-malignant serous tubal intraepithelial carcinoma (STIC), and ultimately lead to HGSOC. Here, we show that the transition from folded, soluble to aggregated mutant p53 occurs during the malignant transformation of benign precursor lesions into HGSOC. We analyzed fallopian tube tissue collected from ten salpingo-oophorectomy cases and determined the proportion of cells carrying soluble versus mis-folded/mutant p53 through conformation-sensitive staining and quantification. Misfolded p53 protein, prone to aggregation, is present in STICs and HG-SOCs, but notably absent from preneoplastic lesions and surrounding healthy tissue. Overall, our results indicate that aggregation of mutant p53 is a structural defect that distinguishes preneoplastic early lesions from late premalignant and malignant ones, offering a potential treatment window for targeting p53 aggregation and halting ovarian cancer progression.

Resistin alleviates lipopolysaccharide-induced inflammation in bovine alveolar macrophages by activating the AMPK/mTOR signaling pathway and autophagy

ObjectiveResistin (RETN) is an adipocyte-specific hormone that participates in metabolism and modulates cellular inflammation. Our study aimed to assess the effects of RETN treatment on autophagy and the underlying molecular and biological mechanisms in bovine alveolar macrophages (BAMs). MethodsThe optimal concentration of RETN + lipopolysaccharide (LPS) on macrophages was screened and then used to co-culture with alveolar macrophages. Autophagosomes in BAMs were examined using a transmission electron microscope (TEM). Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of microtubule-associated protein light chain 3 (LC3) and p62. Western blot (WB) was used to detect the protein expressions of LC3 and p62. The distribution of LC3 and p62 proteins in the cells was observed by immunofluorescence (IF). The concentrations of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expression of adenosine-monophosphate-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), and p-mTOR was detected using WB. ResultsThe treatment of BAMs with RETN or LPS increased the number of autophagosomes and the ratio of LC3II/LC3I and decreased the expression level of p62 protein. RETN treatment significantly triggered autophagy compared to LPS treatment. Moreover, the ratios of p-AMPK/AMPK and p-mTOR/mTOR were upregulated and downregulated, respectively, after RETN treatment, suggesting that AMPK/mTOR signaling pathway activation is required for RETN-mediated autophagy in BAMs. Additionally, the ratio of LC3-II/LC3-I was lower, and the concentrations of IL-1β, IL-6, and TNF-α significantly decreased in the LPS and RETN co-treatment groups compared to the single LPS treatment group. However, both autophagy- and LPS-induced inflammation were partially alleviated by RETN treatment. ConclusionRETN can promote autophagy in BAMs by activating the AMPK/mTOR signaling pathway, it may help prevent LPS-induced inflammation.

Effects of uric acid on oxidative stress in vascular smooth muscle cells.

Hyperuricemia during hypertension is associated with aberrant vascular functions and increased oxidative stress, which affects endothelial dysfunction. Nevertheless, the molecular mechanisms underlying the effects of uric acid on vascular smooth muscle cells (VSMCs) through oxidative stress remain unclear. The aim of the present study was to investigate the dose- and time-dependent effects of uric acid on oxidative stress and p53 protein expression in VSMCs. VSMCs were incubated with various concentrations of uric acid (0-50 mg/dl) for different time periods (1-24 h). Thiobarbituric acid reactive substances (TBARs), protein carbonylation and nitric oxide (NO) levels were determined using appropriate assay kits. Superoxide anion release was detected using the Görlach method. Western blotting was performed to determine the protein expression levels of p53. The findings demonstrated that the application of uric acid led to an increase in protein carbonylation and superoxide anion levels while causing a decrease in NO levels. Conversely, no significant effect was observed on TBARS levels. Additionally, it was observed that high concentrations of uric acid suppressed p53 expression at 6, 12 and 24 h. The present study provided evidence that the influence of uric acid on oxidative stress was more closely associated with time than dose; however, not all effects observed were strictly time-dependent.

Exopolysaccharides from the Green Microalga Strain Coelastrella sp. BGV-Isolation, Characterization, and Assessment of Anticancer Potential.

Algal metabolites have been extensively studied as potential anticancer therapeutics. Among them, polysaccharides have attracted much attention because of their beneficial biological effects and safety. In the present research, the chemical characteristics, antitumor, and proapoptotic activities of extracellular polysaccharides (EPS) isolated from a new Bulgarian strain of the green microalga Coelastrella sp. BGV were investigated. A fast and convenient method of precipitation with cold ethanol was used to isolate EPS from the culture medium. The chemical characteristics of the isolated EPS were examined by colorimetric and spectrophotometric analyses, HPSEC-RID and HPLC-UV chromatography, and FT-IR spectroscopy. The results showed that the isolated EPS sample consists of three carbohydrate fractions with different molecular weights (11.5 × 104 Da, 30.7 × 104 Da, and 72.4 × 104 Da, respectively) and contains 7.14 (w/w%) protein. HPLC-UV analysis revealed the presence of galactose and fucose. The total uronic acid content in the sample was 4.5 (w/w%). The IR-FT spectrum of EPS revealed the presence of various functional groups typical of a polysaccharide (or proteoglycan) composed primarily of neutral sugars. The anticancer potential of the obtained EPS was assessed using cell lines with cancerous and non-cancerous origins as in vitro experimental models. The results of the performed MTT assay showed that EPS reduced the viability of the cervical and mammary carcinoma cell lines HeLa and MCF-7, while the control non-cancer cell lines BALB/3T3 and HaCaT were less affected. The HeLa cell line showed the highest sensitivity to the effects of EPS and was therefore used for further studies of its anticancer potential. The ability of EPS to inhibit cancer cell migration was demonstrated by wound-healing (scratch) assay. The cell cycle FACS analysis indicated that the EPS treatment induced significant increases in the sub G1 cell population and decreases of the percentages of cells in the G1, S, and G2-M phases, compared to the control. The fluorescent microscopy studies performed using three different staining methods in combination with Annexin V-FITC flow cytometric analysis clearly demonstrate the ability of EPS to induce cancer cell death via the apoptosis pathway. Moreover, an altered pattern and intensity of the immunocytochemical staining for the apoptosis- and proliferation-related proteins p53, bcl2, and Ki67 was detected in EPS-treated HeLa cancer cells as compared to the untreated controls. The obtained results characterize the new local strain of green microalgae Coelastrella sp. BGV as a producer of EPS with selective antitumor activity and provide an opportunity for further studies of its pharmacological and biotechnological potential.

Abstract A026: Studying the synergistic potential of a p53 reactivator with ROS-mediated therapies for the treatment of pancreatic cancer

Abstract Introduction: Pancreatic cancer, with a dismal five-year survival rate of just 12%, demands innovative approaches due to its resistance to current therapies. TP53 mutations, present in 75% of patients, make mutant p53 an appealing target. Thus, p53 reactivation, restoring its tumour suppressor functions, represents a promising strategy to address this challenge. While the proposed reactivator has shown efficacy in other malignancies, its potential in pancreatic cancer therapy requires further exploration. Methods: In this project, we have assessed the potential of repurposing a reactivator of mutant p53 protein, to inactivate pancreatic cancer cells. We have performed RNA-sequencing analysis in patient-derived models available in the lab, to study the signalling pathways regulated by the p53 reactivator anticancer activity. We have also studied the possibility to synergistically combine this repurposing strategy with reactive oxygen species (ROS)-mediated therapeutic approaches, as we previously observed p53 reactivation inhibits the glutathione antioxidant pathway. Results: The combination treatment induced synergistic cell death (3-fold increase) compared to monotherapy as evaluated by different molecular assays (e.g. MTT and Live/Death Assay). It also increased ROS levels (Flow cytometry) and induced paraptosis and apoptosis (Western Blot, Tomography Electron Microscopy). The RNA-seq data revealed differential expression of more than 200 genes, confirming the activation of oxidative stress and a cellular response to unfolded proteins. Moreover, Sanger sequencing results demonstrated that the sensibility of each pancreatic model to treatment was dependent on the mutational status of TP53. Conclusion/discussion: The combination therapy of a TP53 reactivator and a ROS mediated therapeutic strategy works synergistically in patient derived models, inducing cell death and changes in the expression of certain RNAs directly involved in unfolded protein response. This pilot study shows that this combination is a feasible strategy to treat pancreatic cancer, setting the path for its testing on in vivo models. Citation Format: Daniel Parra Sánchez, Andrés García-Sampedro, Isabela Laughland, Sylvain Peuget, Galina Selivanova, Stephen P Pereira, Pilar Acedo. Studying the synergistic potential of a p53 reactivator with ROS-mediated therapies for the treatment of pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A026.

GABA(A) Receptor Activation Drives GABARAP-Nix Mediated Autophagy to Radiation-Sensitize Primary and Brain-Metastatic Lung Adenocarcinoma Tumors.

In non-small cell lung cancer (NSCLC) treatment, radiotherapy responses are not durable and toxicity limits therapy. We find that AM-101, a synthetic benzodiazepine activator of GABA(A) receptor, impairs the viability and clonogenicity of both primary and brain-metastatic NSCLC cells. Employing a human-relevant ex vivo 'chip', AM-101 is as efficacious as docetaxel, a chemotherapeutic used with radiotherapy for advanced-stage NSCLC. In vivo, AM-101 potentiates radiation, including conferring a significant survival benefit to mice bearing NSCLC intracranial tumors generated using a patient-derived metastatic line. GABA(A) receptor activation stimulates a selective-autophagic response via the multimerization of GABA(A) receptor-associated protein, GABARAP, the stabilization of mitochondrial receptor Nix, and the utilization of ubiquitin-binding protein p62. A high-affinity peptide disrupting Nix binding to GABARAP inhibits AM-101 cytotoxicity. This supports a model of GABA(A) receptor activation driving a GABARAP-Nix multimerization axis that triggers autophagy. In patients receiving radiotherapy, GABA(A) receptor activation may improve tumor control while allowing radiation dose de-intensification to reduce toxicity.

A PDE1 inhibitor, vinpocetine, ameliorates epithelial-mesenchymal transition and renal fibrosis in adenine-induced chronic kidney injury in rats by targeting the DNMT1/Klotho/β-catenin/Snail 1 and MMP-7 pathways.

Tubulointerstitial fibrosis (TIF) is present with chronic kidney disease (CKD). Vinpocetine (Vinpo) is used for treating cerebrovascular deficits, exhibiting some kidney-beneficial effects; however, its role in TIF is uncertain. So, the aim of this study was to investigate its potential impact on adenine-induced fibrotic CKD and explore the underlying mechanistic aspects. Eighteen male Wistar rats were categorized into three groups (n = 6 each). Group I was kept as controls and given saline; group II received adenine (300 mg/kg, twice weekly, i.p.) for induction of the CKD model; and group III was administered Vinpo (20 mg/kg/d, orally) concurrently with adenine. All treatments were administered for 4 weeks. Vinpo revealed an improvement in renal function and an alleviation of inflammation triggered by adenine via diminishing serum tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels. Further, Vinpo repressed the epithelial-mesenchymal transition (EMT) with preserved E-cadherin mRNA expression and lowered gene and immune expression of fibronectin and vimentin, respectively, besides attenuating the elevated G2/M arrest-related molecules (renal Ki67 protein contents and p21 gene expression). Renal pathological alterations caused by adenine were attenuated upon Vinpo administration. Interestingly, Vinpo suppressed abnormal renal β-catenin immunoreactivity, Snail 1, and MMP-7 gene expression while simultaneously restored Klotho protein expression by downregulating DNA methyltransferase 1 enzyme (DNMT1) protein expression in the kidney. These data indicated that Vinpo effectively mitigated EMT and G2/M arrest-induced renal fibrosis in adenine-induced CKD rats by targeting DNMT1-associated Klotho suppression, subsequently inhibiting β-catenin and its fibrotic downstream genes.

Knockdown of the Clock gene in the liver aggravates MASLD in mice via inhibiting lipophagy.

The increased global prevalence of metabolic dysfunction-associated steatohepatitis (MASLD) has been closely associated with chronic disorders of the circadian clock. Herein, we investigate the role of Clock, a core circadian gene, in the pathogenesis of MASLD. Wild-type (WT) and liver-specific Clock knockdown (Clock-KD) mice were fed a Western diet for 20weeks to induce MASLD. A cellular MASLD model was established by treating AML12 cells with free fatty acids and the effects of Clock knockdown were examined following transfection with Clock siRNA. Increased lipid deposition and more severe steatohepatitis and fibrosis were observed in the livers of Western diet-fed but not normal chow diet-fed Clock-KD mice after 20weeks compared to WT mice. Moreover, the Clock gene was found to be significantly downregulated in WT MASLD mice. The Clock gene was shown to regulate the expression of lipophagy-related proteins (LC3B, P62, RAB7, and PLIN2) in vivo and in vitro. Knockdown of Clock was found to inhibit lipophagy resulting in increased accumulation of lipid droplets in the mouse liver and AML12 cells. Interestingly, the CLOCK protein was shown to interact with P62. However, knockdown of the Clock gene did not promote transcription of the P62 gene but suppressed degradation of the P62 protein during lipophagy in AML12 cells. The hepatic Clock gene regulates lipophagy and affects lipid droplet deposition in liver cells, and thus plays a critical role in the development of MASLD induced by a Western diet.