P53 Protein Expression Research Articles

Patulin induces ROS-dependent cardiac cell toxicity by inducing DNA damage and activating endoplasmic reticulum stress apoptotic pathway

Patulin (PAT) is one of the mycotoxins commonly found in agricultural products and fruits, and has obvious toxic effects on animals and humans. PAT has been found to cause myocardial toxicity and oxidative damage, but the mechanism of myocardial toxicity remained to be elucidated. We investigated the toxic effects and potential mechanisms of PAT on human cardiomyocytes and explored the effects of reactive oxygen species (ROS) on them. The study showed that treatment with PAT for 24 h decreased cell viability and superoxide dismutase (SOD) activity, and increased ROS and lactate dehydrogenase (LDH) levels. Moreover, in addition to detecting increased γ-H2AX expression and observing nuclear damage, the comet assay also showed increased DNA tail distance in the PAT-treated group, followed by an increase in phosphorylation of the p53 protein and p21 protein expression, and a decrease in CDK1 and Cyclin B1 protein expression, and G2/M phase arrest. In addition, PAT induced endoplasmic reticulum stress (ERS) and induced apoptosis, as evidenced by Ca2+ increase, ER enlargement and swelling, and upregulation of ERS-related genes and proteins expression, and increased expression of three apoptotic pathway proteins under ERS, including CHOP, JNK, and caspase-12. Meanwhile, N-acetylcysteine (NAC, a ROS scavenger) reversed the negative effects of PAT treatment on cells. These results clarify that excessive ROS production by PAT-treated AC16 cells not only causes DNA damage, leading to cell cycle arrest, but also causes ERS, which triggers apoptotic pathways to cause apoptosis.

Sulfated polysaccharides of Laetiporus sulphureus fruiting bodies exhibit anti-breast cancer activity through cell cycle arrest, apoptosis induction, and inhibiting cell migration

Ethnopharmacological relevanceLaetiporus sulphureus has long been used as an edible and medicinal mushroom in Asia, America, and Europe. Its fruiting bodies are widely used in folk medicine for treating cancer, gastric diseases, cough, and rheumatism. Polysaccharides are an important bioactive component of mushrooms. In nature, sulfated polysaccharides have never been reported in mushrooms. Furthermore, there is no information on differences in physicochemical properties and anti-breast cancer activities between polysaccharides (PS) and sulfated polysaccharides (SPS) of L. sulphureus. Aim of the studyThis study aimed to investigate the physicochemical properties of PS and SPS isolated from fruiting bodies of L. sulphureus and examine their anti-proliferative effects and mechanism(s) of action on MDA-MB-231 breast cancer cells. MethodsPolysaccharides (PS) were isolated using hot water and ethanol precipitation methods. Sulfated polysaccharides (SPS) were isolated by the papain-assisted hydrolysis method. Physicochemical properties comprising sugar, protein, uronic acid, and sulfate contents, and molecular weight, monosaccharide composition, and structural conformation were analyzed on PS and SPS. In the anti-cancer study, a triple-negative breast cancer cell line (MDA-MB-231) and a normal human mammary epithelial cell line (H184B5F5/M10) were used to evaluate the anti-proliferative activity of PS and SPS, and their mechanism(s) of action. ResultsThe results showed that SPS, which had higher sulfate and protein contents and diversified monosaccharide composition, exhibited more potent anti-proliferative activity against MDA-MB-231 cells than PS. Furthermore, it had a selective cytotoxic effect on breast cancer cells but not the normal cells. SPS induced cell cycle arrest at G0/G1 phase via down-regulating CDK4 and cyclin D1 and up-regulating p21 protein expression. Breast cancer cell apoptosis was not observed until 72 h after SPS treatment. In addition, SPS also markedly inhibited breast cancer cell migration. ConclusionThis study demonstrates that SPS exhibited selective cytotoxicity and was more potent than PS in inhibiting MDA-MB-231 cell proliferation. The contents of sulfate and protein, and monosaccharide composition could be the main factors affecting the anti-breast cancer activity of L. sulphureus SPS.

Investigating the Impact of Phenolic and Terpene Fractions extracted from Prunus arabica on p53 Protein Expression in AMJ13 and SK-GT-4 Human Cancer Cell Lines

Investigating the Impact of Phenolic and Terpene Fractions extracted from Prunus arabica on p53 Protein Expression in AMJ13 and SK-GT-4 Human Cancer Cell Lines

E3 ubiquitin-ligase RNF138 may regulate p53 protein expression to regulate the self-renewal and tumorigenicity of glioma stem cells.

Glioblastoma multiforme (GBM), the most malignant tumor of the central nervous system, is characterized by poor survival and high recurrence. Glioma stem cells (GSCs) are key to treating GBM and are regulated by various signaling pathways. Ubiquitination, a post-translational modification, plays an important regulatory role in many biological processes. Ring finger protein 138 (RNF138) is an E3 ubiquitin-protein ligase that is highly expressed in several tumors; however, its role in GBM is unclear. This study investigated whether RNF138 regulates the self-renewal ability of glioma stem GSCs to treat GBM. The expression of RNF138 in glioma tissues and its correlation with GSCs were analyzed using bioinformatics. Short hairpin ribonucleic acid (RNA) was designed to downregulate the expression of RNF138 in GSCs, and immunofluorescence, secondary pellet formation, and western blotting were used to detect changes in GSC markers and self-renewal ability. The effects of RNF138 on p53 protein expression were determined by immunofluorescence and western blotting. The effects of RNF138 on the self-renewal and tumorigenic abilities of GSCs were evaluated in vivo. RNF138 expression was higher in glioma tissues than in normal brain tissues, and was highly expressed in GSCs. RNF138 downregulation significantly decreased the expression of the GSC markers cluster of differentiation 133 (CD133) and nestin. Mechanistically, RNF138 may interfere with the self-renewal ability of GSCs by regulating the expression of p53. RNF138 downregulation in vivo prolonged survival time and regulated the expression of p53 protein in tumor-bearing mice. RNF138 may regulate the expression of p53 protein through ubiquitination, thereby affecting the self-renewal and tumorigenic ability of GSCs. This study provides a scientific basis for the treatment of glioblastoma by targeting RNF138 to inhibit GSCs.

Effect of Ningxin-Yishen Formula on D-galactose-induced Premature Ovarian Insufficiency Mice by Inhibiting p53

: The prevalence of premature ovarian insufficiency (POI) is gradually increasing, safer and more effective treatments are urgently needed. The aim of this study was to evaluate the effects of Ningxin-Yishen formula (NXYSF) on D -galactose-induced POI mice as well as to shed a light on its potential mechanisms. : Six to eight weeks old C57BL/6 female mice were used in this study and randomly divided into six groups: control group; model group; estradiol valerate (EV) treatment group and NXYSF treatment group with graded doses (9.5, 19, and 38 g∙kg −1 /day). Both EV and NXYSF treatments were initiated at the 15th day of modeling and lasted for 28 days. Afterwards, the ovarian function was evaluated in each group by analyzing the proportion of primordial follicles as well as the serum sex hormone levels. Furthermore, network pharmacology approach was performed to elucidate the potential targets of NXYSF, which were verified through western blotting experiments finally. : NXYSF could significantly reverse the inefficiency of weight gain caused by POI, and promote the development of primordial follicles. In addition, it could restore the abnormal serum anti-Müllerian hormone (AMH), estradiol (E 2 ), luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Moreover, some crucial key gene targets including TP53 were proposed to be related with the effect of NXYSF through network pharmacology analysis. Last but importantly, western blotting experiments confirmed that NXYSF could inhibit the expression of p53 protein in mouse ovary. : Our findings proposed that NXYSF might be an effective alternative treatment for POI.

Diosmin Promotes Myogenesis via Activating the Akt/FOXO1 Pathway to Facilitate the Proliferation of C2C12 Myoblasts.

Our previous study with artificial intelligence (AI)-assisted screening found that diosmin, a natural flavonoid extracted from citrus, may affect myoblast proliferation and differentiation. At present, few studies have been conducted regarding the biological function of diosmin in muscle cells. Here, using molecular biological techniques, we found that diosmin elevated the proliferation ability of C2C12 myoblasts via activating the Akt/FOXO1 pathway to promote FOXO1 nuclear export, thus repressing p27 protein expression, increasing CDK2, CDK4, and cyclin D1 and cyclin E1 protein expression and accelerating cell cycle transformation, which contributed to myogenesis. Moreover, diosmin suppressed differentiation of C2C12 myoblasts by delaying the terminal exit of the cell cycle in early differentiated myoblasts and inhibiting autophagic flux in mature myotubes. Furthermore, diosmin promoted myogenesis by activating the Akt/FOXO1 pathway to facilitate myoblast proliferation, which had a positive biological effect on the repair of muscle injury. This study revealed the effect and mechanism of diosmin on skeletal muscle cells and simultaneously provided a new candidate drug for the treatment of myopathy.

Inhibition of mitochondria induces apoptosis and reduces telomere length and activity in acute myeloid leukemia stem cells.

Acute myeloid leukemia (AML) is a highly lethal hematological malignancy in adults and children. Abnormal proliferation of leukemia stem cells (LSC) with CD34+ and CD38- phenotypes are the main clinical features of AML. Patients with AML face drug resistance and treatment failure due to a default in stem and progenitor cells. Therefore, defining LSC properties is necessary for targeting leukemia-initiating cells. Mitochondrial mass and activity increase in AML initiating cells compared with normal stem cells. This idea has offered the inhibition of the mitochondrial translation machinery to reduce the number of leukemia-initiating cells in patients with AML Tigecycline is an FDA-approved microbial antibiotic that inhibits oxidative phosphorylation in mitochondria, resulting in the suppression of leukemia cell proliferation with little toxicity to normal cells. Thus, the present study was conducted to evaluate whether LSC is influenced by mitochondrial inhibition. We measured the IC50 of tigecycline in KG-1a AML cell lines. KG-1a AML cell lines were separated into CD34+ and CD34- cells by MACS. In the following, these cells were treated with 20 µM (IC50) tigecycline. The expression of Annexin/PI, Caspase 3, apoptotic genes (BCL2, BCLX, BAX, BAD, and P53) and proteins (P53, BAX, BCL2 and Caspase 9) was evaluated in CD34+ , CD34- and KG-1a AML cells. In addition, the telomere length and expression of hTERT were evaluated in this study. The results indicated that BCl2 (gene and protein) and BCLX gene dramatically decreased. In addition, BAD, BAX, and P53 gene and protein expression significantly increased in CD34+ AML cells compared to CD34- AML cells. The results also suggested that tigecycline induced intrinsic (Cleaved-caspase 9/Pro-Caspase 9 ratio) and p53-mediated apoptosis. Furthermore, hTERT gene expression and telomere length decreased in the tigecycline-treated groups. Taken together, our findings indicate that inhibition of mitochondrial activity with tigecycline can induce apoptosis in cancer stem cells and can be used as a novel method for cancer therapy.

Prenylated chromones and flavonoids isolated from the roots of Flemingia macrophylla and their anti-lung cancer activity

BackgroundThe successful launch of icaritin, a therapeutic drug for liver cancer derived from Epimedium brevicornu, has provided new impetus for the development of prenylated flavonoids in the field of oncology. Flemingia macrophylla is reported to contain characteristic prenylated flavonoids which can regulate the p53 protein. We aimed to isolate these constituents and conduct activity evaluation, structure–activity relationship, and mechanism studies to provide candidate compounds for antitumor drug development.MethodsIn this study, chromatographic techniques combined with spectroscopic methods were used to separate, purify, and identify the constituents of Flemingia macrophylla methanol extract. The cytotoxic activity of the constituents was evaluated using an MTT assay with A549 and H1975 cells as the model. The binding mechanism between the compounds and the p53 protein was investigated with molecular docking and validated with cellular thermal shift assay (CETSA). Western blotting (WB) was employed to detect the expression of p53 protein and apoptosis-related proteins in cells.ResultsChiral HPLC separation of racemates 1 and 7 provided two pairs of undescribed enantiomers (1a/1b and 7a/7b), along with eight known compounds (2 − 9) isolated from Flemingia macrophylla roots. Their structures were elucidated by spectroscopic analysis, and the absolute configurations of the enantiomers were determined from experimental and calculated electronic circular dichroism data. Compounds 1 − 7, and the non-prenyl analogues 10 − 13, were evaluated for cytotoxic activity against the human lung cancer A549 and H1975 cell line. Compounds 5 − 7 displayed better cytotoxicity than the positive control icaritin in A549 and H1975, with IC50 values ranging from 4.50 to 19.83 μmol·L-1 and < 5 μmol·L-1, respectively. The structure–activity relationships of the chromone or flavonoid analogues against A549 cells were discussed. Molecular docking results demonstrated that compound 7a has strong interaction with p53 and WB indicated that 7a induced apoptosis by increasing the p53 protein, decreasing the anti-apoptotic protein Bcl-2, and activating the caspase family in A549 cells. These results suggest that prenylated flavonoids are potential p53 protein activators.ConclusionThis study demonstrates that Flemingia macrophylla is rich in prenylated flavonoid constituents, among which compounds 5 and 7 exhibited significant cytotoxic activity against A549 cells and served as reference candidates for the design and development of prenylated compounds as antitumor therapeutic drugs.Graphical

Herbal Melanin Inhibits Real-Time Cell Proliferation, Downregulates Anti-Apoptotic Proteins and Upregulates Pro-Apoptotic p53 Expression in MDA-MB-231 and HCT-116 Cancer Cell Lines.

Background and Objectives: Cancer is the second-most-important deadly disease in the world, leading to severe socioeconomic consequences and posing a public threat. Consequently, breast and colorectal cancers are significant cancer types that affect women and men more commonly, respectively. Treatment failure or recurrent diseases frequently occur due to resistance, in addition to the side effects of the currently available anticancer agents. Therefore, in this study, herbal melanin anticancer activity was investigated against human breast adenocarcinoma (MDA-MB-231) and human colorectal (HCT 116) cell proliferation and the expression of downregulated anti-apoptotic proteins and upregulated pro-apoptotic p53. Materials and Methods: MDA-MB-231 and HCT 116 cells were monitored for their real-time proliferation properties using Xcelligence. Herbal melanin of various concentrations significantly inhibited MDA-MB-231 and HCT 116 cell proliferation. Then, the expression of proapoptotic and anti-apoptotic proteins such as p53, Bcl-2 and Bcl-xl was studied using Western blotting. Results: The Bcl-2 and Bcl-xl expressions were downregulated, while the p53 expression was upregulated after treatment with herbal melanin. Similarly, the expression of apoptotic proteins such as Bcl-2, Bcl-xl, XIAP, Survivin, Bid, Bax, p53, Cytochrome C, PARP genes and mRNA was studied after herbal melanin treatment using real-time PCR, which revealed the downregulation of Bcl-2, Bcl-xl, XIAP and Survivin and the upregulation of Bid, Bax, p53, Cytochrome C and PARP apoptotic protein expression. Also, caspase 3 and 9 expressions were monitored after the treatment with herbal melanin, which revealed the upregulation of both the MDA-MB-231 and HCT 116 cell types. Conclusions: Overall, herbal melanin can be used as an alternative anticancer agent against the MDA-MB-231 and HCT 116 cell types.

RNF144B-mediated p21 degradation regulated by HDAC3 contribute to enhancing ovarian cancer growth and metastasis

We have shown before that HDAC3 was involved in the pathogenesis of ovarian cancer; however, the specific mechanism of HDAC3 on the pathogenesis of ovarian cancer has not been thoroughly studied. To explore the related proteins in the mechanism of HDAC3 on ovarian cancer. The transcriptome profiles were identified in ovarian carcinoma cells with HDAC3 knockdown or overexpression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis were used to verify transfection efficiency. Immunofluorescence staining were performed to detect the expression levels of HDAC3 and RNF144B in tissues. Cell proliferation, apoptosis, migration and invasion were confirmed by cell counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase (TUNEL) and transwell assay, respectively. The protein expression of p53, p21, Bax and Bcl-2 was confirmed by western blot, and CoIP assay was used to validate RNF144B/P21/P53 interaction. Meanwhile, the protein synthesis inhibitor cycloheximide (CHX) was performed to treat cells to probe p21 stability. Finally, we established an in vivo tumor model to explore the effects of HDAC3 and RNF144B on tumor growth. Microarray results showed that among the overlapping genes in the two profiles (HDAC3 knockdown and overexpression), RNF144B was decreased or increased in ovarian carcinoma cells with HDAC3 knockdown or overexpression, HDAC3 overexpression promoted RNF144B expression, and HDAC3 knockdown hindered RNF144B levels. The levels of HDAC3 and RNF144B in malignant ovarian cancer were significantly higher than those in normal ovarian tissue and benign ovarian cancer tissue. RNF144B promoted cell proliferation, migration and invasion, and inhibited cell apoptosis. In addition, overexpression of HDAC3 or RNF144B inhibited p53/p21/Bax expression and promoted Bcl-2 expression. Knockout of HDAC3 or RNF144B has the opposite effect, and RNF144B interacted with p21 and regulated the p21/p53 complex degradation, and finally in vivo experiments proved that HDAC3 and RNF144B promoted tumor growth. RNF144B-mediated p21 degradation regulated by HDAC3 contributed to enhancing ovarian cancer growth and metastasis.

MiR-20b-5p involves in vascular aging induced by hyperhomocysteinemia

Hyperhomocysteinemia (HHcy) is an independent risk factor of atherosclerosis (AS). Some reports have shown that homocysteine (Hcy) could accelerate the development of AS by promoting endothelial cell senescence. miRNAs were widely involved in the pathophysiology of HHcy. However, few studies have focused on the changes of miRNA-mRNA networks in the artery of HHcy patients. For this reason, RNA-sequencing was adopted to investigate the expression of miRNA and mRNA in HHcy model mouse arteries. We found that the expression of 216 mRNAs and 48 miRNAs were significantly changed. Using TargetScan and miRDB web tools, 29 miRNA-mRNA pairs were predicted. Notably, miR-20b-5p and FJX1 shared the highest predicted score in TargetScan, and further study indicated that the miR-20b-5p inhibitor significantly upregulated the FJX1 expression in HHcy human umbilical vein endothelial cells (HUVECs) model. PPI analysis revealed an important sub-network which was centered on CDK1. Gene ontology (GO) enrichment analysis showed that HHcy had a significant effect on cell cycle. Further experiments found that Hcy management increased reactive oxygen species (ROS) generation, the activity of senescence associated β-galactosidase (SA-β-gal) and the protein expression of p16 and p21 in HUVECs, which were rescued by miR-20b-5p inhibitor. In general, our research indicated the important role of miR-20b-5p in HHcy-related endothelial cell senescence.

MTOR Inhibitor Rapalink-1 Prevents Ethanol-Induced Senescence in Endothelial Cells.

The cardiovascular risk factors, including smoking, ethanol, and oxidative stress, can induce cellular senescence. The senescent cells increase the expression and release of pro-inflammatory molecules and matrix metalloproteinase (MMPs). These pro-inflammatory molecules and MMPs promote the infiltration and accumulation of inflammatory cells in the vascular tissue, exacerbating vascular tissue inflammation. MMPs damage vascular tissue by degenerating the extracellular matrix. Consequently, these cellular and molecular events promote the initiation and progression of cardiovascular diseases. We used Rapalink-1, an mTOR inhibitor, to block ethanol-induced senescence. Rapalink-1 inhibited oxidative-stress-induced DNA damage and senescence in endothelial cells exposed to ethanol. It attenuated the relative protein expression of senescence marker P21 and improved the relative protein expression of DNA repair protein KU70 and aging marker Lamin B1. It inhibited the activation of NF-κB, MAPKs (P38 and ERK), and mTOR pathway proteins (mTOR, 4EBP-1, and S6). Moreover, Rapalink-1 suppressed ethanol-induced mRNA expression of ICAM-1, E-selectin, MCP-1, IL-8, MMP-2, and TIMP-2. Rapalink-1 also reduced the relative protein expression of MMP-2. In summary, Rapalink-1 prevented senescence, inhibited pro-inflammatory pathway activation, and ameliorated pro-inflammatory molecule expression and MMP-2.

Finasteride effectively attenuates the impairments of left ventricular function and cardiac sympathovagal balance in both aging and obese male rats via reducing systemic oxidative stress

Abstract Background Cardiovascular diseases are commonly found in the elderly and obese population manifested by left ventricular (LV) dysfunction and impaired cardiac sympathovagal balance. Finasteride (FIN), a 5-alpha reductase inhibitor (5-ARI), is a universal prescription for patients with benign prostatic hyperplasia (BPH). Since BPH is commonly found in obesity and aging, it is essential to investigate whether FIN could have an impact on the LV function and cardiac sympathovagal balance in either elderly or obese conditions. Purpose We tested the hypothesis that impaired LV function and cardiac sympathovagal balance are observed in aging and obese conditions, and FIN improves these parameters through a reduction of systemic oxidative stress in both aging and obese male rats. Methods Twenty-four male Wistar rats (aged 8 weeks old) were randomly divided into 3 groups (n=8/group) to receive one of the followings: 1) normal diet (ND) feeding, 2) ND feeding with D-galactose injection (150 mg/kg/day, subcutaneous injection, 18 weeks) for creating an aging model, and 3) HFD feeding for 18 weeks for creating an obesity model. At week 13, rats in each group were divided into 2 subgroups (n=4/subgroup) and received either a vehicle (drinking water) or FIN (5 mg/kg/day) via oral gavage. At the end of treatment, echocardiography was performed to assess LV function, and the heart rate variability (HRV) was determined to quantify cardiac sympathovagal balance. The blood was collected, and serum was used to determine serum malondialdehyde (MDA) levels as a marker of systemic oxidative stress. Results At week 13, D-galactose injection successfully induced aging as indicated by increasing cardiac p53 protein expression. HFD also caused obesity in those rats as indicated by increased body weight and visceral fat weight. LV dysfunction (as indicated by reduced %LVEF and %LVFS) and cardiac sympathovalgal imbalance (as indicated by an increased LF/HF ratio) were observed in both models (Figure 1A-C). This functional impairment was associated with an increased MDA levels (Figure 1D). Treatment with FIN successfully reduced cardiac sympathovagal imbalance in both models (Figure 1C); however, an improvement of LV function was found only in aging rats-treated with FIN (Figure 1A, B). Consistent with a reduction of cardiac sympathovagal imbalance, FIN decreased systemic oxidative stress in both models (Figure 1D). Conclusions FIN effectively improved cardiac sympathovagal balance in rats under both aging and obese conditions. However, FIN reduced LV dysfunction only in aging rats. Interestingly, FIN did not affect these cardiac parameters in normal healthy rats. These findings suggest the cardioprotective potential of FIN in both aging and obese populations.

Effect of IFN‑γ encapsulated liposomes on major signal transduction pathways in the lymphocytes of patients with lung cancer.

Globally, lung cancer affected 2.2 million individuals and caused 1.8 million deaths in 2021. Lung cancer is caused by smoking, genetics and other factors. IFN-γ has anticancer activity. However, the mechanism by which IFN-γ has an effect on lung cancer is not fully understood. The present study aimed to assess the effect of IFN-γ on the peripheral lymphocytes of patients with lung cancer compared with healthy controls. The efficacy of IFN-γ against oxidative stress was assessed using a comet repair assay and the effects of IFN-γ on p53, PARP1 and OGG1 genes and protein levels in lymphocytes was evaluated by RT-qPCR and western blotting. DNA damage was significantly reduced in the lymphocytes of patients treated with IFN-γ. However, there was no effect in the cells of healthy individuals after treatment with naked IFN-γ [IFN-γ (N)] and liposomal IFN-γ [IFN-γ (L)]. Following treatment with IFN-γ (N) and IFN-γ (L), the p53, PARP1 and OGG1 protein and gene expression levels were significantly increased (P<0.001). It has been suggested that IFN-γ may induce p53-mediated cell cycle arrest and DNA repair in patients. These findings supported the idea that IFN-γ (N) and IFN-γ (L) may serve a significant role in the treatment of lung cancer, via cell cycle arrest of cancer cells and repair mechanisms.

Effects of the Supercritical Fluid Extract of Magnolia figo on Inducing the Apoptosis of Human Non-Small-Cell Lung Cancer Cells

Lung cancer has a high incidence rate worldwide, necessitating the development of new drugs. Although Magnolia figo (Lour.) DC. is known for its medicinal properties, studies on its efficacy against lung cancer are lacking. This study investigated whether the supercritical fluid extract of M. figo (FMO) can induce apoptosis in A549, a human non-small-cell lung cancer cell line. The cell viability was assessed using an MTT assay. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis and flow cytometry analysis were conducted. The expression of factors was assessed through Western blotting analyses. Gas chromatography–mass spectrometry (GC-MS) was performed. The results revealed that FMO treatment exhibited cytotoxicity, demonstrating dose-dependent effects. The TUNEL analysis and flow cytometry analysis revealed that FMO induced apoptosis in A549 cells. The Western blotting analysis revealed that FMO upregulated the expression of p53 and Bax protein, and downregulated the expression of Bcl-2 protein. The GC-MS analysis revealed eight components identified in FMO. These findings indicate that FMO can induce A549 apoptosis through the p53/Bcl-2/Bax pathways, confirming the apoptotic effects of M. figo on lung cancer cells. These results highlight the potential, for the first time, of M. figo as a source for developing novel drugs for lung cancer treatment.

Loss of p73 Expression Contributes to Chronic Obstructive Pulmonary Disease.

Rationale: Multiciliated cell (MCC) loss and/or dysfunction is common in the small airways of patients with chronic obstructive pulmonary disease (COPD), but it is unclear if this contributes to COPD lung pathology. Objectives: To determine if loss of p73 causes a COPD-like phenotype in mice and explore whether smoking or COPD impact p73 expression. Methods: p73floxE7-E9 mice were crossed with Shh-Cre mice to generate mice lacking MCCs in the airway epithelium. The resulting p73Δairway mice were analyzed using electron microscopy, flow cytometry, morphometry, forced oscillation technique, and single-cell RNA sequencing. Furthermore, the effects of cigarette smoke on p73 transcript and protein expression were examined using invitro and invivo models and in studies including airway epithelium from smokers and patients with COPD. Measurements and Main Results: Loss of functional p73 in the respiratory epithelium resulted in a near-complete absence of MCCs in p73Δairway mice. In adulthood, these mice spontaneously developed neutrophilic inflammation and emphysema-like lung remodeling and had progressive loss of secretory cells. Exposure of normal airway epithelium cells to cigarette smoke rapidly and durably suppressed p73 expression invitro and invivo. Furthermore, tumor protein 73 mRNA expression was reduced in the airways of current smokers (n = 82) compared with former smokers (n = 69), and p73-expressing MCCs were reduced in the small airways of patients with COPD (n = 11) compared with control subjects without COPD (n = 12). Conclusions: Loss of functional p73 in murine airway epithelium results in the absence of MCCs and promotes COPD-like lung pathology. In smokers and patients with COPD, loss of p73 may contribute to MCC loss or dysfunction.

Cooperatively inhibition effect of miR-143-5p and miR-145-5p in tumorigenesis of glioblastoma cells through modulating AKT signaling pathway

Introduction: As the most common aggressive primary brain tumor, glioblastoma is inevitably a recurrent malignancy whose patients’ prognosis is poor. miR-143 and miR-145, as tumor suppressor miRNAs, are downregulated through tumorigenesis of multiple human cancers, including glioblastoma. These two miRNAs regulate numerous cellular processes, such as proliferation and migration. This research was intended to explore the simultaneous replacement effect of miR-143, and miR-145 on in vitro tumorgenicity of U87 glioblastoma cells. Methods: U87 cells were cultured, and transfected with miR-143-5p and miR-145-5p. Afterward, the changes in cell viability, and apoptosis induction were determined by MTT assay and Annexin V/PI staining. The accumulation of cells at the cell cycle phases was assessed using the flow cytometry. Wound healing and colony formation assays were performed to study cell migration. qRT-PCR and western blot techniques were utilized to quantify gene expression levels. Results: Our results showed that miR-143-5p and 145-5p exogenous upregulation cooperatively diminished cell viability, and enhanced U-87 cell apoptosis by modulating Caspase-3/8/9, Bax, and Bcl-2 protein expression. The combination therapy increased accumulation of cells at the sub-G1 phase by modulating CDK1, Cyclin D1, and P53 protein expression. miR-143/145-5p significantly decreased cell migration, and reduced colony formation ability by the downregulation of c-Myc and CD44 gene expression. Furthermore, the results showed the combination therapy of these miRNAs could remarkably downregulate phosphorylated-AKT expression levels. Conclusion: In conclusion, miR-143 and miR-145 were indicated to show cooperative anti- cancer effects on glioblastoma cells via modulating AKT signaling as a new therapeutic approach.

Alcoholic beer consumption permutes P21 and cyclin D1 expression, oxidative stress factors, and histomorphometric parameters in rat testis

BackgroundEthanol consumption is increasingly prevalent in communities and has several side effects for humans. Chronic alcohol consumption is often associated with decreased libido and infertility. This study aimed to evaluate the impact of beer on spermatogenesis, proteins, and gene expression of P21 and cyclin D1 in testicular tissue. MethodTwenty-four male mice were assigned to 4 groups. The control group received normal saline, and the experimental groups received alcoholic beer (3 g/kg BW as 20% v/v). After 7, 15, and 35 days, mice were sacrificed, and a part of testicular tissues was stored at −70 °C to measure the protein and gene expression of P21 and cyclin D1 by Western blot and real-time PCR. Moreover, this assay measured the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT). Histomorphometry and histopathology were done. ResultsThe consumption of alcoholic beer led to increased protein and gene expression of P21 and decreased expression of cyclin D1, as well as reduced levels of SOD and CAT, increased levels of MDA, and subsequent tissue damage in the testicular tissue in a time-dependent manner. Also, the diameter of seminiferous tubules and the thickness of the germinal epithelium were reduced in a time-dependent manner, and the percentage of seminiferous tubules with tubular differentiation, replacement, and negative spermiogenesis coefficients increased significantly. ConclusionAlcoholic beer disrupted the process of cell division in the testes by reducing the expression of cyclin D1, increasing p21 expression, and inducing oxidative stress, which in turn reduced sperm production and quality.

AZD-7648, a DNA-PK Inhibitor, Induces DNA Damage, Apoptosis, and Cell Cycle Arrest in Chronic and Acute Myeloid Leukemia Cells.

The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML (HEL, HL-60, KG-1, NB-4, and THP-1) cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor.

Lymphovascular invasion and p16 expression are independent prognostic factors in stage I vulvar squamous cell carcinoma

The objective of this study was to identify clinicopathologic parameters associated with disease outcome in FIGO stage I vulvar squamous cell carcinoma (vSqCC). The cohort consisted of 126 patients diagnosed with vSqCC in the period 2006–2016 who underwent primary vulvar surgery and evaluation of groin lymph node status. Tumors were reviewed by an experienced gynecologic pathologist. p16 and p53 protein expression by immunohistochemistry and HPV status were analyzed in 116 tumors. Clinicopathologic parameters, protein expression and HPV status were analyzed for association with progression-free and overall survival (PFS, OS). p16 expression and aberrant p53 were found in 49 (42%) and 61 (53%) tumors, respectively. Sixty-six tumors were HPV-associated (57%). Relapse was diagnosed in 35/126 (28%) of patients, and 23 (18%) died of disease. Tumor diameter > 4 cm (p = 0.013), lymphovascular space invasion (LVSI; p < 0.001), the presence of lichen sclerosus (p = 0.019), p16 expression (p = 0.007), p53 expression (p = 0.012), HPV status (p = 0.021), lymph node metastasis (p < 0.001) and post-operative radiotherapy (p < 0.001) were significantly related to OS in univariate analysis. Tumor diameter > 4 cm (p = 0.038), LVSI (p = 0.003), the presence of lichen sclerosus (p = 0.004), p16 expression (p = 0.004), HPV status (p = 0.039), lymph node metastasis (p < 0.001) and post-operative treatment (p < 0.001), were significantly related to PFS in univariate analysis. Age, BMI and surgical resection involvement were not significantly associated with OS or PFS. In multivariate Cox analysis, LVSI and p16 expression were independent prognosticators of OS (p < 0.001 and p = 0.02, respectively) and PFS (p = 0.018, p = 0.037). In conclusion, LVSI and p16 expression are independent prognostic factors in stage I vSqCC.