Expression Of P53 Proteins Research Articles

Clinicopathological correlations in 38 cases of gastroenteropancreatic high-grade neuroendocrine neoplasms

ObjectiveDiagnosis and treatment of gastroenteropancreatic high-grade neuroendocrine neoplasms (GEP-HG-NENs), particularly G3 well-differentiated neuroendocrine tumours (NETs) and poorly differentiated neuroendocrine carcinomas (NECs) relies on histopathological morphology, immunohistochemistry, and molecular biological markers, which are lacking especially in cases with ambiguous histomorphology. In this study to contribute to the development of more targeted treatment strategies, we examined various immunohistochemical and molecular biological markers and their association with clinicopathological features in GEP-HG-NENs.MethodsWe included 38 patients with GEP-HG-NENs in this study, with their retrospective follow-up data. The expression of tumour protein p53 (TP53), RB transcriptional corepressor 1 (RB1), somatostatin receptor 2 (SSTR2), clusterin (CLU), and marker of proliferation Ki-67 (MKI67) was immunohistochemically analysed. KRAS proto-oncogene, GTPase (KRAS) and B-Raf proto-oncogene, serine/threonine kinase (BRAF) V600E expression was evaluated using quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between immunohistochemical and molecular biological markers and clinicopathological characteristics were examined using a Cox risk regression model, receiver operating characteristic (ROC) curve, and Kaplan–Meier survival analyses.ResultsSSTR2, RB, TP53, and CLU expression differed between NET G3 and NECs, with variations among the NET G3 and small- and large-cell NEC (SCNEC and LCNEC, respectively) groups (p < 0.05). The median MKI67 proliferative index was approximately 40% and 70% in G3 NETs and NECs, respectively. The NET G3 group exhibited a median survival of 25 months, indicating a relatively better prognosis than that of the NECs group (median survival, 11 months). Both Kaplan–Meier survival analysis and the Cox risk regression model indicated a statistical correlation among treatment methods, CLU expression, and prognosis (p < 0.05). The BRAF V600E mutation rate was 32.4% in G3 NETs and SCNEC, demonstrating a significant difference between both types (p = 0.0086). Furthermore, ROC curve analysis highlighted the diagnostic significance of the positive expression of the immunohistochemical markers CLU, SSTR2, and RB in identifying NET G3.ConclusionTo guide more suitable treatment strategies, it is essential to develop and apply valuable and more targeted immunohistochemical and molecular pathological markers for a comprehensive analysis.

MELATONIN ENHANCES TEMOZOLOMIDE-INDUCED APOPTOSIS IN GLIOBLASTOMA AND NEUROBLASTOMA CELLS.

The combination of temozolomide (TMZ) and paclitaxel (PTX) is the most commonly used chemotherapy regimen for glioblastoma, but there is no specific treatment for neuroblastoma due to the acquired multidrug resistance. Approximately half of treated glioblastoma patients develop resistance to TMZ and experience serious side effects. Melatonin (MEL), a multifunctional hormone long known for its antitumor effects, has a great advantage in combination cancer therapy thanks to its ability to affect tumors differently than normal cells. This study aims to evaluate the in vitro inhibitory effects of MEL in combination with TMZ on cancer cell viability and to elucidate the underlying mechanisms in the glioblastoma and neuroblastoma cell lines. C6 (Rattus norvegicus) and N1E-115 (Mus musculus) cancer cell lines and C8-D1A (mice) healthy cell lines were used. Cell proliferation was evaluated using the MTT test. IC50 values were determined by probit analysis. Two concentrations of TMZ (IC50 and 1/2 IC50) were used to induce cytotoxicity in the C6 and N1E-115 cell lines, both alone and in combination with PXT and MEL (all at IC50). The viable, dead, and apoptotic cells were determined by image-based cytometry using Annexin V/PI staining. The gene expression related to signaling pathways was assessed by the quantitative reverse transcription polymerase chain reaction (qRT-PCR), and key proteins were identified by the Western blot analysis. MTT assay showed that the combination of TMZ and MEL significantly reduces the viability of both glioblastoma and neuroblastoma cells compared to the vehicle-treated controls. Notably, MEL combined with 1/2 IC50 TMZ showed a significant death rate of cancer cells compared to controls and PTX. According to qRT-PCR data, the TMZ + MEL combination resulted in the upregulation of the genes of antioxidative enzymes (Sod1 and Sod2) and DNA repair genes (Mlh1, Exo1, and Rad18) in both cell lines. Moreover, the levels of Nfkb1 and Pik3cg were significantly reduced following the TMZ + MEL treatment. The combination of MEL with TMZ also enhanced the cell cycle arrest and increased the expression of p53 and pro-apoptotic proteins (Bax and caspase-3), while significantly decreasing the expression of anti-apoptotic protein Bcl-2. Our findings indicate that the combination of MEL with a low dose of TMZ may serve as an upstream inducer of apoptosis. This suggests the potential development of a novel selective therapeutic strategy as an alternative to TMZ for the treatment of both glioblastoma and neuroblastoma.

Association between the levels of toxic heavy metals and schizophrenia in the population of Guangxi, China: A Case-Control Study

The relationship between body levels of heavy metals and the risk of schizophrenia remains unclear. This study investigates the relationship between plasma levels of toxic heavy metals and the risk of schizophrenia among adults in Guangxi, China. Plasma concentrations of lead (Pb), cadmium (Cd), arsenic (As), and chromium (Cr) were measured using inductively coupled plasma mass spectrometry (ICP-MS). To evaluate both the single and combined effects of metal exposure on the risk of schizophrenia, we employed multivariate logistic regression, Bayesian Kernel Machine Regression (BKMR), and generalized Weighted Quantile Sum (gWQS) models. Additionally, we employed the Comparative Toxicogenomics Database (CTD) to analyze the mechanistic pathways through which metal mixtures may induce schizophrenia. Relative mRNA expression levels were measured using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to predict potential biological functions. In logistic regression models, compared to the lowest exposure group (Q1), the odds ratios (ORs) for Pb in groups Q2, Q3, and Q4 were 2.18 (95% CI: 1.20 - 3.94), 4.74 (95% CI: 2.52 - 8.95), and 3.62 (95% CI: 1.80 - 7.28), respectively. Both BKMR and gWQS models indicated a positive correlation between the combined effects of toxic heavy metal mixtures and the risk of schizophrenia, with Pb demonstrating the most substantial impact, particularly in older adults and females. Elevated levels of tumor necrosis factor (TNF) and interleukin-1 beta (IL-1β) were observed in patients with schizophrenia, while the expression of tumor protein p53 (TP53) was significantly reduced. These findings underscore the critical need to avoid exposure to toxic heavy metals to prevent schizophrenia, highlighting significant public health implications.

Apigenin Ameliorates H2O2-Induced Oxidative Damage in Melanocytes through Nuclear Factor-E2-Related Factor 2 (Nrf2) and Phosphatidylinositol 3-Kinase (PI3K)/Protein Kinase B (Akt)/Mammalian Target of Rapamycin (mTOR) Pathways and Reducing the Generation of Reactive Oxygen Species (ROS) in Zebrafish

Background: Apigenin is one of the natural flavonoids found mainly in natural plants, as well as some fruits and vegetables, with celery in particular being the most abundant. Apigenin has antioxidant, anti-tumor, anti-inflammatory, and anticancer effects. In this research, we attempted to further investigate the effects of apigenin on the mechanism of repairing oxidative cell damage. The present study hopes to provide a potential candidate for abnormal skin pigmentation disorders. Methods: We used 0.4 mM H2O2 to treat B16F10 cells for 12 h to establish a model of oxidative stress in melanocytes, and then we gave apigenin (0.1~5 μM) to B16F10 cells for 48 h, and detected the expression levels of melanin synthesis-related proteins, dendritic regulation-related proteins, antioxidant signaling pathway- and Nrf2 signaling pathway-related proteins, autophagy, and autophagy-regulated pathways by immunoblotting using Western blotting. The expression levels of PI3K/Akt/mTOR proteins were measured by β-galactosidase staining and Western blotting for cellular decay, JC-1 staining for mitochondrial membrane potential, and Western blotting for mitochondrial fusion- and mitochondrial autophagy-related proteins. Results: Apigenin exerts antioxidant effects by activating the Nrf2 pathway, and apigenin up-regulates the expression of melanin synthesis-related proteins Tyr, TRP1, TRP2, and gp100, which are reduced in melanocytes under oxidative stress. By inhibiting the expression of senescence-related proteins p53 and p21, and delaying cellular senescence, we detected the mitochondrial membrane potential using JC-1, and found that apigenin improved the reduction in mitochondrial membrane potential in melanocytes under oxidative stress, and maintained the normal function of mitochondria. In addition, we further detected the key regulatory proteins of mitochondrial fusion and division, MFF, p-DRP1 (S637), and p-DRP1 (S616), and found that apigenin inhibited the down-regulation of fusion-associated protein, p-DRP1 (S637), and the up-regulation of division-associated proteins, MFF and p-DRP1 (S616), due to oxidative stress in melanocytes, and promoted the mitochondrial fusion and ameliorated the imbalance between mitochondrial division and fusion. We further detected the expression of fusion-related proteins OPA1 and Mitofusion-1, and found that apigenin restored the expression of the above fusion proteins under oxidative stress, which further indicated that apigenin promoted mitochondrial fusion, improved the imbalance between mitochondrial division and fusion, and delayed the loss of mitochondrial membrane potential. Apigenin promotes the expression of melanocyte autophagy-related proteins and the key mitochondrial autophagy proteins BNIP3L/Nix under oxidative stress, and activates the PINK1/Parkin signaling pathway by up-regulating the expression of autophagy-related proteins, as well as the expression of PINK1 and Parkin proteins, to promote melanocyte autophagy and mitochondrial autophagy. Conclusions: Apigenin exerts anti-melanocyte premature aging and detachment effects by promoting melanin synthesis, autophagy, and mitochondrial autophagy in melanocytes, and inhibiting oxidative cell damage and senescence.

The Potential Anticancer Potency of Kolaviron on Colorectal Adenocarcinoma (Caco-2) Cells.

Globally, colorectal cancer (CRC) is categorized as the third type of cancer associated with mortalities. Chemotherapeutic drugs such as cisplatin can be used to treat cancer-affected patients. However, several adverse effects are associated with its application. This motivated the researchers to search for alternatives that are more efficient and have fewer undesirable effects. Kolaviron is a bioflavonoid that has been reported to have antioxidant and anti-inflammatory properties. This study aimed to compare the anticancer effects of kolaviron and cisplatin on Caco-2 cells. The IC50 of kolaviron and cisplatin were calculated, and redox status, apoptotic-related proteins and the cell cycle were also examined. Caco-2 cells were treated with kolaviron ( ⅓, and ½ of IC50 dose) and cisplatin (IC50 dose) for 24 h and 48 h. Cell viability was assessed using the MTT protocol. Redox status and apoptotic-related proteins, in addition to the cell cycle, were examined. The MTT assay showed the IC50 of kolaviron is 9.49 μg/mL, and that of cisplatin is 2.71 μg/ml against Caco-2 cells. Further, both doses of kolaviron significantly increased the leakage of lactate dehydrogenase (LDH), the production of reactive oxygen species (ROS), and lipoperoxidation (LPO), besides decreasing the antioxidant potency of tumor cells as revealed by the diminished reduced glutathione (GSH). At the molecular level, a significant increase in the levels of p53, cytochrome c, Bax, and caspase 3 was recorded, coupled with a decrease in the level of Bcl2, after treating the Caco-2 cells with kolaviron and cisplatin. Furthermore, kolaviron demonstrated asserted more effects on apoptosis and increased cell percentage in the subG1 phase. In addition, a notable decrease in the expression of proliferating cell nuclear antigen (PCNA) and cyclin D1 is associated with an increase in the expression of tumor protein P53 (TP53) in kolaviron-treated Caco-2 cells cancerous cells. Conclusively, these data suggest that kolaviron has a potential antitumor capacity against colorectal cancer via multiple pathways, including enhancement of ROS production, redox status, p53 pathway, and apoptosis. Therefore, this study authenticated the capability of kolaviron as a valuable chemotherapeutic agent.

Protective effect of Lonicerae japonicae flos extract against doxorubicin-induced liver injury in mice

To explore the mechanism underlying the protective effect of Lonicerae japonicae flos (LJF) extract against doxorubicin (DOX) -induced liver injury in mice. Network pharmacology methods were used to obtain the intersection genes between LJF targets and disease targets, based on which the protein-protein interaction (PPI) network was constructed using STRING database for screening the core targets using Cytoscape software. DAVID database was used for bioinformatics analysis, and the core components and core targets were verified using molecular docking study. In a mouse model of DOX-induced liver injury, the effect of LJF extract on liver pathologies, serum levels of ALT and AST, and hepatic expressions of HYP, ROS, TNF-α, IL-6, COL-Ⅳ and P53 proteins were evaluated using HE and Masson staining, ELISA, and Western blotting. We identified 12 core targets from 43 intersection genes involving cancer pathway, IL-17 signaling pathway, and TNF signaling pathways. Molecular docking study suggested that 10 core components of LJF could bind to different core targets. The mice with DOX-induced liver injury showed elevated serum AST and ALT levels with obvious liver injury and fibrosis, increased ROS content, and enhanced expressions of TNF-α, IL-6, HYP, COL-Ⅳ and P53 proteins in the liver tissue. All these changes in the mouse models were significantly alleviated by treatment with LJF extract, suggesting obviously lowered levels of oxidative stress, inflammation and fibrosis in the liver tissues. LJF extract is capable of alleviating DOX-induced liver injury in mice by downregulating Trp53, TNF and IL-6 to reduce liver oxidative stress, inflammation and fibrosis.

Decreased expression of p53 is associated with down expression of zyxin in breast cancer.

Breast cancer (BC) is considered one of the most common malignant tumors leading to death in women, and genetic factors have a crucial role in BC pathogenesis. Zyxin (ZYX) is one of these factors that may be important in p53 level and function. Thus, the present work aimed to investigate the ZYX gene and protein expression in tumor tissue and matched margin tissue and its correlation with the p53 expression. In a present case-control study, 30 tumors and 30 matched margin tissues were obtained from Iran Tumor Bank/Tehran University of Medical Sciences. Real-time polymerase chain reaction and western blot analysis techniques were applied to evaluate the genes and protein expression, respectively. The data showed that expression of the ZYX gene in tumor tissues significantly decreased (p = 0.0274) compared to matched margin tissues. In contrast, the p53 gene expression in tumor tissues had no significant difference with matched margin tissues. Additionally, we observed that ZYX and p53 genes expression in tumor tissues of estrogen receptor-positive patients had significant elevation than estrogen receptor-negative patients (p < 0.001, p < 0.001, respectively). The data of the western blot analysis technique showed that protein expression of ZYX (p = 0.0024) and P53 protein (p = 0.0218) in tumor tissues was significantly reduced compared to matched margin tissues. Additionally, our analysis showed a direct and significant correlation between the expression of ZYX and p53 proteins (r = 0.7797, p = 0.0126) and expression of ZYX and p53 genes (r = 0.3079, p = 0.0187). Based on our observation, ZYX might have a tumor suppressor role and is associated with p53.

5-aminolevulinic acid photodynamic therapy protects against UVB-induced skin photoaging: A DNA-repairing mechanism involving the BER signalling pathway.

Low-dose 5-aminolevulinic acid photodynamic therapy (ALA-PDT) has been used to cope with skin photoaging, and is thought to involve DNA damage repair responses. However, it is still unknown how low-dose ALA-PDT regulates DNA damage repair to curb skin photoaging. We established a photoaging model using human dermal fibroblasts (HDFs) and rat skin. RNA-sequencing (RNA-seq) analysis was conducted to identify differentially expressed genes (DEGs) in HDFs before and after low-dose ALA-PDT treatment, followed by bioinformatics analysis. Senescence-associated β-galactosidase (SA-β-gal) staining was employed to assess skin aging-related manifestations and Western blotting to evaluate the expression of associated proteins. A comet assay was used to detect cellular DNA damage, while immunofluorescence to examine the expression of 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in cells and skin tissues. In both invivo and invitro models, low-dose ALA-PDT alleviated the manifestations of ultraviolet B (UVB)-induced skin photoaging. Low-dose ALA-PDT significantly reduced DNA damage in photoaged HDFs. Furthermore, low-dose ALA-PDT accelerated the clearance of the photoproduct 8-oxo-dG in photoaged HDFs and superficial dermis of photoaged rat skin. RNA-seq analysis suggested that low-dose ALA-PDT upregulated the expression of key genes in the base excision repair (BER) pathway. Further functional validation showed that inhibition on BER expression by using UPF1069 significantly suppressed SA-β-gal activity, G2/M phase ratio, expression of aging-associated proteins P16, P21, P53, and MUTYH proteins, as well as clearance of the photoproduct 8-oxo-dG in photoaged HDFs. Low-dose ALA-PDT exerts anti-photoaging effects by activating the BER signalling pathway.

Mannan-binding lectin inhibits oxidative stress-induced senescence via the NAD+/Sirt1 pathway

Prolonged or excessive oxidative stress can lead to premature cellular and body aging. Mannan-binding lectin (MBL) is synthesized by the liver and plays an important role in innate immunity, anti-inflammation, and anti-oxidation, and has a positive impact on health and longevity. To date, few studies investigated the role of MBL in attenuating oxidative stress-induced senescence. In this study, we evaluated the role of MBL in oxidative stress-induced premature aging and explored its underlying mechanism in C57BL/6 mice and mouse embryonic fibroblasts (NIH/3T3). First, we established an oxidative premature senescence model induced by D-galactose in C57BL/6 mice. We found that MBL-deficient mice had a marked aging-like appearance, reduced learning and spatial exploration abilities, severe liver pathological damage, and significantly upregulated expression of Senescence-associated proteins (p53 and p21), inflammatory kinesins (IL-1β and IL-6), and the senescence β-galactosidase (SA-β-Gal) positive rate as compared with WT mice. In the H2O2-induced oxidative senescence model of NIH/3T3 cells, consistent results were obtained after MBL intervention. In addition, MBL effectively inhibited G1 phase arrest, ROS levels, DNA damage, and mitochondrial dysfunction in premature senescent cells. Mechanistically, we found that oxidative stress inhibited the nicotinamide adenine dinucleotide (NAD+)/ silent information regulator 1 (Sirt1) signaling pathway, while MBL activated the NAD+/Sirt1 signaling pathway inhibited by oxidative stress. In addition, MBL could activate the NAD+/Sirt1 pathway by upregulating NAMPT, which in turn inhibited p38 phosphorylation by activating the NAD+/Sirt1 pathway. In conclusion, MBL inhibits oxidative aging, which may facilitate the development of therapeutics to delay oxidative aging.

Cytotoxicity of alkaloids isolated from Peganum harmala seeds on HCT116 human colon cancer cells.

The present study aimed to elucidate the potential anticancer activity and mechanism of P. harmala's alkaloid extract, harmine (HAR), and harmaline (HAL) in HCT-116 colorectal cancer cells. P. harmala's alkaloid was extracted from harmala seeds. HCT-116 cells were treated with P. harmala's alkaloid extract, HAR and HAL. Cytotoxicity was determined by MTT assay, apoptotic activity detected via flow cytometry and acridine orange (AO)/ethidium bromide (EB) dual staining, and cell cycle distribution analyzed withflow cytometry. The mRNA expression of Bcl-2-associated X protein (Bax) and glycogen synthase kinase-3 beta (GSK3β) was measured by real-time PCR. Furthermore, the expression of Bax, Bcl-2, GSK3β and p53 proteins, were determined by western blotting. The findings indicated that, P. harmala's alkaloids extract, HAR and HAL were significantly cytotoxic toward HCT116 cells after 24 and 48h of treatment. We showed that P. harmala's alkaloid extract induce apoptosis and cell cycle arrest at G2 phase in the HCT116 cell line. Downregulation of GSK3β and Bcl-2 and upregulation of Bax and p53 were observed. The findings of this study indicate that the P. harmala's alkaloid extract has anticancer activity and may be further investigated to develop future anticancer chemotherapeutic agents.

Particulate matter exposure induces adverse effects on endometrium and fertility via aberrant inflammatory and apoptotic pathways in vitro and in vivo

This study aimed to evaluate the adverse effects of particulate matter (PM) exposure on endometrial cells and fertility and to identify possible underlying mechanisms. Thirteen women (aged 15–52 years) were included in this study. Enrolled patients underwent laparoscopic surgery at Gangnam Severance Hospital between 1 January and 31 December 2021. For in vivo experiments, 36 female and nine male C57BL/6 mice were randomly divided into control(vehicle), low-dose(10 mg/kg/d), and high-dose exposure groups(20 mg/kg/d). PM was inhaled nasally for four weeks and natural mating was performed. NIST® SRM® 1648a was used for PM exposure. qRT-PCR, western blotting and Masson's trichrome staining were performed. PM treatment in human endometrial stromal cells induced inflammation with significant upregulation of IL-1β, p–NF–kB, and p-c-Jun compared to those of controls. Additionally, PM treatment significantly increased apoptosis in human endometrial stromal cells by downregulating p-AKT and upregulating p-p53/p53, Cas-3, BAX/Bcl-2, p-AMPK, and p-ERK. After PM treatment, the relative expression of IL-1β, IL-6, TNF-α, p–NF–κB, p-c-Jun, and p-Nrf2/Nrf2 significantly increased in murine endometrium compared to those of the controls. Expression of apoptotic proteins p53, p27, and Cas-3, was also significantly elevated in murine endometrium of the PM exposure group compared to that of the controls. A significant increase in expression of procollagen Ⅰ, and Masson's trichrome staining scores in the murine endometrium was noted after PM treatment. PM treatment significantly decreased ERα expression. After natural mating, all 3 female mice in the control group gave birth to 25 offspring (mean 8.1), whereas in the low-dose PM treatment group, two of three female mice gave birth to nine offspring (mean 4.5). No pregnant mice or offspring was present in the high-dose PM treatment group. PM exposure induces adverse effects on the endometrium through aberrant activation of inflammatory and apoptotic pathways and is associated with detrimental effects on murine fertility.

The role and mechanism of engineered nanovesicles derived from hair follicle mesenchymal stem cells in the treatment of UVB-induced skin photoaging.

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are effective in the treatment of skin photoaging; however, their low yield and functional decline with passage progression limit their clinical application. Cell-derived nanovesicles (CNVs) are potential alternatives that can address the limitations of EVs derived from MSCs and are conducive to clinical transformations. Hair follicle mesenchymal stem cells (HFMSCs), a type of MSCs, have demonstrated the function of repairing skin tissues; nevertheless, the efficacy of CNVs from HFMSCs (HFMSC-CNVs) in the treatment of skin photoaging remains unclear. Therefore, ultraviolet radiation B (UVB)-induced photoaging nude mice and human dermal fibroblasts (HDFs) were used as experimental models to investigate the therapeutic effects of HFMSC-CNVs in photoaging models. HFMSC-CNVs were successfully prepared using the mechanical extrusion method. UVB-induced nude mice and HDFs were used as experimental models of photoaging. Multiple approaches, including hematoxylin-eosin and Masson staining, immunohistochemistry, immunofluorescence, detection of reactive oxygen species (ROS), flow cytometry, western blotting, and other experimental methods, were combined to investigate the possible effects and mechanisms of HFMSC-CNVs in the treatment of skin photoaging. In the nude mouse model of skin photoaging, treatment with HFMSC-CNVs reduced UVB-induced skin wrinkles (p < 0.05) and subcutaneous capillary dilation, alleviated epidermis thickening (p < 0.001), and dermal thinning (p < 0.001). Furthermore, HFMSC-CNVs upregulated proliferating cell nuclear antigen (PCNA) expression (p < 0.05) and decreased the levels of ROS, β-galactosidase (β-Gal), and CD86 (p < 0.01). Invitro experiments, treatment with HFMSC-CNVs enhanced the cellular activity of UVB-exposed HDFs (p < 0.05), and reduced ROS levels and the percentage of senescent cells (p < 0.001), and alleviated cell cycle arrest (p < 0.001). HFMSC-CNVs upregulated the expression of Collagen I (Col I), SMAD2/3, transforming growth factor beta (TGF-β), catalase (CAT), glutathione peroxidase-1 (GPX-1), and superoxide dismutase-1 (SOD-1) (p < 0.05) and downregulated the expression of cycle suppressor protein (p53), cell cycle suppressor protein (p21), and matrix metalloproteinase 3 (MMP3) (p < 0.05). Conclusively, the anti-photoaging properties of HFMSC-CNVs were confirmed both invivo and invitro. HFMSC-CNVs exert anti-photoaging effects by alleviating cell cycle arrest, decreasing cellular senescence and macrophage infiltration, promoting cell proliferation and extracellular matrix (ECM) production, and reducing oxidative stress by increasing the activity of antioxidant enzymes.

Methylthioadenosine phosphorylase and p16 as surrogate diagnostic markers for CDKN2A homozygous deletion in brain tumors

Objective: To examine whether immunohistochemistry of methylthioadenosine phosphorylase (MTAP) and p16 could be used to predict the CDKN2A status in various brain tumors. Methods: A total of 118 cases of IDH-mutant astrocytomas, 16 IDH-wildtype glioblastoma, 17 polymorphic xanthoastrocytoma (PXA) and 20 meningiomas diagnosed at Xuanwu Hospital, Capital Medical University, Beijing, China from November 2017 to October 2023 were collected and analyzed. The CDKN2A status was detected by using fluorescence in situ hybridization or next-generation sequencing. Expression of MTAP and p16 proteins was detected with immunohistochemistry. The association of loss of MTAP/p16 expression with CDKN2A homozygous/heterozygous deletion was examined. Results: Among the 118 cases of IDH-mutant astrocytoma, 13 cases showed homozygous deletion of CDKN2A. All of them had no expression of MTAP while 9 cases had no expression of p16. Among the 16 cases of IDH wild-type glioblastoma, 6 cases showed homozygous deletion of CDKN2A. All 6 cases had no expression of MTAP, while 3 of these cases had no expression of p16 expression. Among the 17 PXA cases, 4 cases showed homozygous deletion of CDKN2A, and the expression of MTAP and p16 was also absent in these 4 cases. Among the 20 cases of meningiomas, 4 cases showed homozygous deletion of CDKN2A. Their expression of MTAP and p16 was also absent. Among the four types of brain tumors, MTAP was significantly correlated with CDKN2A homozygous deletion (P<0.05), with a sensitivity of 100%. However, it was only significantly correlated with the loss of heterozygosity (LOH) of CDKN2A in astrocytomas (P<0.001). P16 was associated with CDKN2A homozygous deletion in IDH-mutant astrocytoma and PXA (P<0.001), but not with the LOH of CDKN2A. Its sensitivity and specificity were lower than that of MTAP. Conclusions: MTAP could serve as a predictive surrogate for CDKN2A homozygous deletion in adult IDH-mutant astrocytoma, PXA, adult IDH-wildtype glioblastoma and meningioma. However, p16 could only be used in the first two tumor types, and its specificity and sensitivity are lower than that of MTAP.

Effect of Chitosan on Synovial Membrane Derived Cells and Anterior Cruciate Ligament Fibroblasts.

Previously, chitosan reduces the senescence-related phenotypes in human foreskin fibroblasts through the transforming growth factor beta (TGF-β) pathway, and enhances the proliferation and migration capabilities of these cells are demonstrated. In this study, we examined whether the senescence-delaying effect of chitosan could be applied to primary knee-related fibroblasts, such as human synovial membrane derived cells (SCs) and anterior cruciate ligament fibroblasts (ACLs). These two types of cells were obtained from donors who needed ACL reconstruction or knee replacement. We found that chitosan treatment effectively reduced aging-associated β-galactosidase (SA-β-gal)-positive cells, downregulated the expression of senescence-related proteins pRB and p53, and enhanced the 5-bromo-2'-deoxyuridine (BrdU) incorporation ability of SCs and ACLs. Moreover, chitosan could make SCs secret more glycosaminoglycans (GAGs) and produce type I collagen. The ability of ACLs to close the wound was also enhanced, and the TGF-β and alpha smooth muscle actin (αSMA) protein expression decreased after chitosan treatment. In summary, chitosan not only delayed the senescence but also enhanced the functions of SCs and ACLs, which is beneficial to the application of chitosan in cell expansion in vitro and cell therapy.

P300 reduces TUBB4B expression to facilitate the biological process of migration and invasion of non-small cell lung cancer cells

This article explored the mechanism of E1A binding protein p300 (P300) and beta-tubulin 4B isotype-encoding gene (TUBB4B) in regulating the migration and invasion of non-small cell lung cancer (NSCLC) cells. TUBB4B and P300 expression in NSCLC tissues and cells was monitored by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. TUBB4B function on NSCLC cell migration, invasion and epithelial-mesenchymal transition (EMT) was monitored by wound healing assay, Transwell experiment and Western blot. The regulation of P300 on TUBB4B was monitored by qRT-PCR and Western blot. Mechanism of P300 and TUBB4B in regulating NSCLC cell migration and invasion was explored by rescue experiment. A xenograft tumor model was established by using nude mouse. As a result, low TUBB4B expression and high P300 expression was discovered in NSCLC tissues and cells. TUBB4B and P300 expression showed a negative correlation in NSCLC tissues. Lower TUBB4B but higher P300 was observed in tumor tissues of NSCLC patients with metastasis. TUBB4B overexpression suppressed NSCLC cell migration, invasion and EMT. TUBB4B silencing had opposite results. P300 overexpression inhibited TUBB4B expression, and P300 silencing facilitated TUBB4B overexpression in NSCLC cells. TUBB4B overexpression counteracted the promotion of P300 overexpression on NSCLC cell invasion and migration. TUBB4B silencing abrogated the inhibition of P300 knockdown on NSCLC cell invasion and migration. TUBB4B overexpression suppressed NSCLC cell in vivo growth. Thus, TUBB4B could be reduced by P300 in NSCLC. It exerted suppression role on NSCLC cell migration, invasion and EMT. TUBB4B may be a novel target for NSCLC treatment.

The molecular profile people of different ages in «normal» and with dilated cardiomyopathy

Background: Dilated cardiomyopathy (DCMP) is a cardiovascular pathology promising for consideration as a clinical model of premature aging of the body. According to the literature, sirtuins-1, 2, 3, 6; melatonin and its receptors (MT-1 and MT-2); markers of cellular aging (proteins p16, p21) and apoptosis (BAX) may have clinical significance for assessing premature aging of the body on the DCMP model. A comparative study of the expression of these molecules in people of different ages, depending on the presence of DCMP, has not been conducted before. The aim of the study: To identify and compare molecular profiles by the level of expression of the studied signaling molecules in the buccal epithelium and autopsy material of the myocardium in people of different ages with the absence of cardiovascular diseases and the presence of dilated cardiomyopathy. Materials and methods: A study of buccal epithelium (174 people) and autopsy material of myocardium (175 people) in patients aged 45-89 years for the expression of signaling molecules: sirtuins 1, 2, 3, 6, proteins p16, p21 and BAX, was conducted. Methods of immunocytochemical and immunohistochemical studies were used. Groups of middle, elderly and senile age with DCMP and without a history of cardiovascular pathology were identified. Results: Characteristic changes in the molecular profile (the level of the studied signaling molecules) in the buccal epithelium associated with age and the presence of DCMP were revealed: in middle–aged patients – a decrease in the expression of sirtuins 3 and 6 by 2,2-2,3 times, melatonin by 1,8 times and its receptors by 1,4-2,5 times; in elderly patients – a decrease in the expression of sirtuins 1 and 3 by 2,5 times; sirtuin 2 by 2,3 times; melatonin and its receptors by 1,4-1,8 times; in elderly patients – a decrease in the expression of sirtuins 3 and 6 by 3,0-4,7 times; sirtuins 1 and 2 by 6,7-7,3 times; melatonin by 2,8 times; MT-1 – 4,1 times and MT-2 – 23,7 times. An increase in the expression of p16 and BAX proteins by 0,6-0,8 times in the buccal epithelium has additional diagnostic information in the diagnosis of DCMP in individuals of different age groups. There is a correlation between changes in the expression of these molecules in the buccal epithelium and the autopsy material of the myocardium. Conclusion: Molecular profiles in people of different age groups, determined by the level of expression of the studied signaling molecules, significantly differ depending on the presence of DCMP. It is characteristic that the direction of changes in the buccal epithelium is similar to the corresponding molecular profile in the autopsy material of the myocardium.

Ochratoxin A (OTA) causes intestinal aging damage through the NLRP3 signaling pathway mediated by calcium overload and oxidative stress.

Ochratoxin A (OTA) is a widespread environmental toxin that poses a serious threat to human and animal health. OTA has been shown to cause cellular and tissue damage and is a global public health problem. However, the effects of OTA on gastrointestinal aging have not been reported. The aim of this study was to investigate the effects of OTA on intestinal aging in vitro and in vivo. In vitro experiments showed that OTA induced cellular inflammation through calcium overload and oxidative stress, significantly up-regulated the expression of P16, P21, and P53 proteins, markedly increased senescence-associated β-galactosidase activity (SA-β-gal) positive cells, and obviously decreased the expression of proliferating cell nuclear antigen (PCNA) proteins, which led to intestinal cell senescence. Meanwhile, we found that treatment with β-carotene ameliorated OTA-induced intestinal cell senescence. Consistent with the results of the in vitro experiments, in vivo studies showed that the intestinal aging of mice fed OTA was significantly higher than that of the control group. In conclusion, OTA may induce intestinal aging through calcium overload, oxidative stress and inflammation. This study lays a foundation for further research on the toxicological effects of OTA.

Abstract 1823: In vitro evaluation of novel pyrimidine nucleosides for the treatment of pancreatic cancer

Abstract The purpose of the study is to synthesize and characterize novel 5-FU analogs (AGY1 and AGY2) and investigate their anticancer activity against pancreatic cancer cell lines. Methods: 5-FU derivatives were synthesized and characterized using Nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC), and elemental analysis to determine the presence of new bond formation and the purity of the derivatives or analogs. 2D cell viability, 3D models, cell migration, and cell cycle studies were performed on MiaPaCa-2 (MP-2) cells and PANC-1 cells. Apoptosis was performed on PANC-1 cells by staining with Acridine Orange and Ethidium Bromide. The metabolic stability of the analogs was determined by using human liver microsomes. PARP, p53, and BAX expressions in PANC-1 were determined by Western Blot. Results: NMR data showed the conjugation of alkyl and tetrahydrofuran groups to the 5-FU ring confirming the formation of AGY1 and AGY2. The elemental analysis of AGY1 and AGY2 showed 99.6% and 99.5 % purity respectively. The IC50 value of the AGY1 against 2D MP-2 was found to be 12.2 ± 1 µM, while the IC50 value of AGY2 was determined to be 2.9 ± 0.2 µM. Both AGY1 and AGY2 anticancer activities were significantly higher than 5-FU (18.5 ± 1.1 µM). However, AGY2 was more effective compared to AGY1. A similar trend was observed for AGY2 analog where the IC50 value for treated 2D PANC-1 was 3.1 ± 0.29 µM and demonstrated a higher anticancer activity compared to AGY1 (6.4 ± 0.38 µM) or 5-FU (7.5 ± 1.1 µM). For cell migration studies, AGY2 (43 ± 5.5) exposed MP-2 cells showed significant arrest towards the wound gap compared to AGY1 (74 ± 18.5) and 5-FU (86 ± 15.6). A similar result of cell migration arrests AGY2 was observed in PANC-1 treated-AGY1, AGY2, and 5-FU. Organoid and spheroid models demonstrated high and comparable anticancer activity of AGY1 and AGY2 compared to 5-FU treated organoid and spheroid models. The cell-cycle analysis showed that AGY2-treated MP-2 cells at 1μΜ concentration showed a higher G1 population arrest (64.25%) compared to AGY1 (54.2%) and 5-FU (55.75%). Similarly, in PANC-1 cells, AGY2 at 1μΜ concentration showed a higher S-phase arrest (53.86%) compared to AGY1 (50.76%) and 5-FU (36.14%). Metabolic stability data showed higher stability of AGY2 (95% intact) compared to AGY1 (85% intact) compared to 5-FU (55% intact) after 2 hour of human liver microsomal incubation. The expressions of p53 and BAX proteins in PANC-1 cells were significantly upregulated and PARP was downregulated at higher concentration when treated with AGY1 and AGY2 compared to 5-FU. Conclusion: We demonstrate that AGY2 showed more promising anticancer activity compared to AGY1 and 5-FU against pancreatic cancer cells. AGY2 has the potential to improve 5-FU’s metabolic stability and enhance its therapeutic efficacy. Citation Format: Raviteja Bulusu, Joy Okoro, Esther Frimpong, Bo Han, Sherise Rogers, Xue Zhu, Edward Agyare. In vitro evaluation of novel pyrimidine nucleosides for the treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1823.

Cytotoxic Activities and Expression of Protein p53 and Bcl-2 Extract and Fraction of Inggu Leaf (Ruta angustifolia [L.] Pers) to T47D Breast Cancer Cells

Inggu leaves are plants that have been traditionally used as an alternative to cancer treatment. The aim of this study was to determine the cytotoxic activity of extracts, water fraction, ethyl acetate fraction and inggu leaf n-hexane fraction (Ruta angustifolia [L.] Pers) on T47D cells and to determine the effect of protein expression of P53 and Bcl-2 genes on inggu leaf active fraction (Ruta angustifolia [L.] Pers). Extract was obtained through maceration method with ethanol 96% solvent. Ethanol extract was then fractionated with liquid-liquid partition. Cytotoxic tests were performed using T47D breast cancer cells and Vero cells with the MTT assay test method and read the absorbance on ELISA reader. To determine the effect of expression of p53 and Bcl-2, an immunocytochemistry test was carried out. The results showed that the n-hexane extract and fraction of inggu leaves had cytotoxic activity against T47D breast cancer cells with IC50 values of 54,210 and 84,118 µg/mL, while the fraction of water and ethyl acetate fractions had no cytotoxic activity with IC50 value &gt; 100 µg / ml. Inggu leaf extract (Ruta angustifolia [L.] Pers) is able to increase the expression of p53 and inhibit the expression of Bcl-2 at concentrations of 30,19-45,54 µg/mL and n-hexane fraction can increase the expression of p53 and can inhibit the expression of Bcl-2 at concentration 49,96-75,82 µg/mL.

Isolation and Identification of a Tibetan Pig Porcine Epidemic Diarrhoea Virus Strain and Its Biological Effects on IPEC-J2 Cells.

Porcine epidemic diarrhoea virus (PEDV) is a coronavirus that can cause severe watery diarrhoea in piglets, with high morbidity and mortality rates, seriously hindering the healthy development of the global swine industry. In this study, we isolated a strain of PEDV from Tibetan pigs and named it CH/GS/2022. Subsequently, we screened the apoptosis signals of PEDV-infected IPEC-J2 cells and studied the correlation between apoptosis signals and cell apoptosis. The results showed that different infections of PEDV induced different degrees of apoptosis in cells, and PEDV-induced cell apoptosis was dose-dependent. We then detected the expression of the p53, p38, JNK, Bax, and Bcl-2 genes in the apoptosis signal pathway. The results showed that 24 h after PEDV infection, the expression of the p53, p38, JNK, and Bax genes in IPEC-J2 cells increased significantly, while the expression of the Bcl-2 gene decreased significantly (p < 0.05). Subsequently, we used Western blot to detect the protein levels of these five genes, and the results showed that PEDV infection upregulated the expression of p53, p38, JNK, and Bax proteins (p < 0.05) while downregulating the expression of Bcl-2 protein (p < 0.05). Thus, it was initially inferred that PEDV infection could regulate cell apoptosis by activating the p53, p38, and JNK signalling pathways. Finally, we further investigated the apoptosis of the cells through the use of inhibitors. The results indicated that the p53 inhibitor Pifithrin-α has a significant inhibitory effect on the expression of the p53 protein after PEDV infection and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p53 is involved in PEDV-induced cell apoptosis. Similarly, the p38 MAPK inhibitor SB203580 has an inhibitory effect on the expression of the p38 protein and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p38 is also involved in PEDV-induced cell apoptosis. On the other hand, the JNK inhibitor SP600125 has no inhibitory effect on the expression of the JNK protein after PEDV infection, but the expression levels of Bax and Bcl-2 proteins have changed. Furthermore, it is noteworthy that SP600125 can inhibit the activity of apoptotic proteins but not their levels, resulting in reduced cell apoptosis. These preliminary results indicated that JNK may be involved in PEDV-induced IPEC-J2 cell apoptosis.