Protein Expression Levels Of P53 Research Articles

SP-8356: A Novel Verbenone Derivative Exerts In Vitro Anti-Non-Small Cell Lung Cancer Effects, Promotes Apoptosis via The P53/MDM2 Axis and Inhibits Tumor Formation in Mice.

Non-small cell lung cancer (NSCLC) stands as a prominent contributor to cancer-related fatalities on a global scale, necessitating the search for novel therapeutic agents. SP-8356, a derivative of (1S)-(-)-verbenone, has shown promise as an anticancer agent in preclinical studies. However, specific mechanisms underlying its effects in NSCLC remain to be elucidated. The aim of this research was to explore the in vitro anti-NSCLC effects of SP-8356, elucidate its mechanisms of action, and assess its efficacy in inhibiting tumor formation in a murine model. In this experimental study, NSCLC cell lines were treated with various concentrations of SP- 8356. Cell viability and proliferation were assessed using MTT and colony formation assays, respectively. Cell cycle distribution was analyzed by flow cytometry, and apoptosis was evaluated by determining apoptotic protein expression. Western blot analysis was conducted to assess protein expression levels of the both p53 and MDM2. Additionally, we evaluated efficacy of the SP-8356 in inhibiting tumor formation of the nude mouse model. SP-8356 demonstrated a concentration-dependent inhibition of cell proliferation in the NSCLC cell lines. Flow cytometric analysis showed that SP-8356 led to cell cycle arrest at the G2/M phase, indicating its potential influence on regulating the cell cycle. SP-8356 treatment was associated with the downregulation of CDK1 and Cyclin B1. Additionally, SP-8356 significantly enhanced apoptosis in NSCLC cells. SP-8356 treatment was associated with the downregulation of Bcl-2, while Bax expression was upregulated. Mechanistically, SP-8356 led to accumulation of the p53 protein levels within the NSCLC cells. This accumulation was mediated through inhibition of its negative regulator, MDM2. Using a nude mouse model demonstrated that SP-8356 effectively inhibited tumor formation in vivo. Our findings shed light on the molecular mechanisms underlying anticancer activity of SP-8356 and highlight its potential as a promising therapeutic candidate for NSCLC treatment.

The inhibition of FTO attenuates the antifibrotic effect of leonurine in rat cardiac fibroblasts

BackgroundMyocardial fibrosis (MF) is a common pathological condition in cardiovascular diseases that often causes severe cardiac dysfunction. MF is characterized by changes in cardiomyocytes, cardiac fibroblasts (CFs), levels of collagen (Col) -1, -3, and overdeposition of the extracellular matrix. Our previous research showed that leonurine (LE) effectively inhibits collagen synthesis and differentiation of CFs, but the mechanism is not fully elucidated. Recent evidence indicates that fat mass and obesity-associated proteins (FTO) regulates the occurrence and development of MF. This study aimed to explore the role of FTO in the antifibrotic effects of LE. MethodsNeonatal rat CFs were isolated, and induced using angiotensin II (Ang II) to establish a cell model of MF. Cell viability, wound healing and transwell assays were used to detect cell activity and migration ability. The protein and mRNA levels of MF-related factors were measured following stimulation with Ang II and LE under normal conditions or after FTO knockdown. The RNA methylation level was measured by dot blot assay. ResultsThe results showed that LE (20, 40 μM) was not toxic to normal CFs. LE reduced the proliferation, migration and collagen synthesis of Ang II-induced CFs. Further investigation showed that FTO was downregulated by Ang II stimulation, whereas LE reversed this effect. FTO knockdown facilitated the migration of CFs, upregulated the protein levels of Col-3, α-SMA and Col-1 in Ang II and LE-stimulated CFs, and enhanced the fluorescence intensity of α-SMA. Furthermore, LE reduced N6-methyladenosine (m6A) RNA methylation, which was partially blocked by FTO knockdown. FTO knockdown also reduced the expression levels of p53 protein in Ang II and LE-stimulated CFs. ConclusionsOur findings suggest that the inhibition of FTO may attenuate the antifibrotic effect of LE in CFs, suggesting that FTO may serve as a key protein for anti-MF of LE.

The mechanism of Semen Persicae-Flos Carthami in treating hypertrophic scar: A study based on network pharmacological analysis and invitro experiments.

Traditional medicine believes that hypertrophic scar (HS) falls into the category of "blood stasis". Chinese herbs for promoting blood circulation and removing blood stasis, activating meridians, and relieving pain are usually selected to treat HS by traditional Chinese medicine (TCM). Both Semen Persicae (SP) and Flos Carthami (FC) are confirmed to be effective for HS. Clinically, SP and FC are often used in combination with each other. However, the pharmacodynamic mechanism and molecular target of SP-FC in the treatment of HS are still unclear. Therefore, this study is intended to explore the mechanism and target of SP-FC in the treatment of HS through network pharmacology combined with invitro cell and molecular biology experiments. Target genes of SP-FC were obtained from the traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP), and targets of HS-related diseases were searched from databases such as Disgenet and GeneCards. Based on the targets searched and obtained, a Venn diagram was plotted to acquire common targets of SP-FC-HS. Next, STRING 11.0 was employed for protein-protein interaction (PPI) network analysis of common targets; and cytoscape 3.9.0 for connection relationship analysis of PPI and plotting of a "drug-component-target" network diagram. Besides, a modified explant culture method was applied to separate primary hypertrophic scar fibroblasts (HSFs); MTT assay to detect cell viability of HSFs after treatment by SP-FC for 24 h; Annexin V-FITC/PI double staining combined with flow cytometry to test apoptosis; western blot to check the protein expression level of p53; and real-time fluorescence quantitative PCR to determine mRNA level of p53. In the analysis of network pharmacology, 269 pharmacological targets of SP, 449 pharmacological targets of FC, and 2569 targets of HS-related diseases were screened from the databases. After plotting the Venn diagram, 116 common targets of SP-FC-HS were acquired. In vitro experiments showed that the expression of p53 in HSFs was decreased. SP-FC significantly reduces the viability of HSFs, increases p53 levels in HSFs, and promotes apoptosis. SP-FC can reduce scar formation by promoting p53 expression.

Human Umbilical Cord Mesenchymal-Stem-Cell-Derived Extracellular Vesicles Reduce Skin Inflammation In Vitro

The protective roles of extracellular vesicles derived from human umbilical cord mesenchymal stem cells against oxazolone-induced damage in the immortalized human keratinocyte cell line HaCaT were investigated. The cells were pretreated with or without UCMSC-derived extracellular vesicles 24 h before oxazolone exposure. The pretreated UVMSC-EVs showed protective activity, elevating cell viability, reducing intracellular ROS, and reducing the changes in the mitochondrial membrane potential compared to the cells with a direct oxazolone treatment alone. The UCMSC-EVs exhibited anti-inflammatory activity via reducing the inflammatory cytokines IL-1β and TNF-α. A mechanism study showed that the UCMSC-EVs increased the protein expression levels of SIRT1 and P53 and reduced P65 protein expression. It was concluded that UVMSC-EVs can induce the antioxidant defense systems of HaCaT cells and that they may have potential as functional ingredients in anti-aging cosmetics for skin care.

Effect of Long Non-Coding RNA LINC01268 on the Malignant Biological Behaviors of Acute Myeloid Leukemia Cells

To investigate the effect of long non-coding RNA LINC01268 on apoptosis of acute myeloid leukemia (AML) cells and related mechanisms. The expression levels of LINC01268 and miR-217 in peripheral blood samples from AML patients and AML cell lines HL-60 and KG-1 were detected by qRT-PCR. HL-60 cells were divided into pcDNA3.1-NC, pcDNA3.1-LINC01268, si-NC, si-LINC01268, miR-NC, miR-217 mimics, si-LINC01268 + inhibitor-NC and si-LINC01268+ miR-217 inhibitor groups. The mRNA expressions of LINC01268 and miR-217 were detected by qRT-PCR. The targeting relationship between LINC01268 and miR-217 was detected by dual-luciferase reporter assay. Cell viability was detected by CCK-8 assay. Cell cycle distribution and apoptosis were detected by flow cytometry. The expression of cell cycle and apoptosis-related proteins p21, Bcl-2, Bax, caspase-3 and PI3K/AKT signaling pathway-related proteins were detected by Western blot. The expression of LINC01268 in peripheral blood samples of AML patients and AML cell lines HL-60 and KG-1 was increased (P < 0.05), and the expression of miR-217 was decreased (P < 0.05). Compared with si-NC group and miR-NC group, the viability of HL-60 cells was decreased in si-LINC01268 group and miR-217 mimics group (P < 0.05), the proportion of cells in G1 phase and apoptosis rate were increased (P < 0.05), the protein expression levels of p21, Bax and caspase-3 were increased (P < 0.05), while the protein expression level of Bcl-2 was decreased (P < 0.05). LINC01268 targeted and negatively regulated the expression of miR-217, and inhibiting the expression of miR-217 partially reversed the effects of LINC01268 interference on the viability, cell cycle and apoptosis of HL-60 cells. Interference with LINC01268 could inhibit the activity of PI3K/AKT signaling pathway. Inhibiting the expression of miR-217 could partially reverse the inhibition of LINC01268 interference on PI3K/AKT signaling pathway. LINC01268 is highly expressed and miR-217 is lowly expressed in AML cells. LINC01268 can promote the activity of PI3K/AKT signaling pathway, increase the survival rate and inhibit the apoptosis of AML cells by targeting miR-217 expression.

Curcumin Enhances Ibrutinib Induced Killing Effect Against TP53-Mutated Chronic Lymphocytic Leukemia Cells in Vitro

Introduction TP53 mutation and del (17p) are the most important poor prognostic factors in patients with Chronic Lymphocytic Leukemia (CLL).Patients with deletion 17p or TP53 mutations tend to have lower response rates to standard first-line chemoimmunotherapy and a more aggressive clinical course(Stilgenbauer et. al. Blood 2014). RESONATE-2 study long-term follow-up showing that ibrutinib continues to demonstrate significant and durable clinical benefit in older patients, including those with TP53 mutation(Barr et. al. Blood Adv 2022).However, CLL bearing del(17p) showed inferior OS and time to next treatment compared to non-del(17p)(Mato et. al. Haematologica 2022). A significant proportion of patients treated with single-agent ibrutinib experienced CLL progression.BTK and/or PLCG2 mutations were present in 80.0% of patients progressing with CLL, confirming prior reports on the high prevalence of these mutations in patients with ibrutinib-resistant CLL(Ahn et. al. Blood 2017). Curcumin is a bioactive natural polyphenol compound extracted from the Curcuma longa(Zingiberaceae plant). Curcumin has anti-tumor effect and can induce apoptosis and inhibit the proliferation of a series of tumor cells(Willenbacher et. al. Int J Mol Sci 2019). Curcumin may limit the development of therapy-related toxicity and reduce the financial burden of treatment.In this study, we found that Curcumin inhibits TP53-mutated CLL cells, and eliciting a strong synergistic cytotoxic effect in combination with ibrutinib. Methods The inhibitory effect of ibrutinib combined with curcumin on the proliferation of TP53-mutated Chronic Lymphocytic Leukemia cells was detected by CCK8, and the apoptosis of TP53-mutated CLL cells induced by ibrutinib combined with curcumin was detected by flow cytometry;The differences of gene and signal pathway expression among different treatment groups were analyzed by RNA-seq ;Using Western blot technology to verify the expression of related pathway proteins and explore the mechanism of curcumin increasing the killing effect of ibrutinib against TP53-mutated Chronic Lymphocytic Leukemia cells. Results The CCK8 results showed that the IC50 of ibrutinib for 24 hours was 40.61±2.35μM, while the IC50 of curcumin for 24 hours was 26.6±0.94μM. The IC50 of ibrutinib for 48 h was 14.6 ±0.8307μM, and the IC50 of curcumin for 48 h was 15.7±2.53μM.Cell inhibition of various group for 48h is shown in Figure 1.The CI of ibrutinib combined with curcumin at 24 hours and 48 hours was calculated by CompuSyn software. The results showed that the CI values of ibrutinib combined with curcumin were less than 1 at 24 h and 48 h, suggesting that ibrutinib combined with curcumin has a synergistic killing effect on CLL cells with TP53 mutation;Flow cytometry showed that the IC50 of ibrutinib for 24 hours was 67.20±2.86μM, while the IC50 of curcumin for 24 hours was 144±6.6 μM. The CI value of ibrutinib combined with curcumin was less than 1 at 24 h, suggesting that ibrutinib combined with curcumin has a synergistic promoting apoptosis on CLL cells with TP53 mutation;RNA-seq analysis and KEGG analysis of down-regulated genes of combination group compared with the control group were mainly enrich in PI3K/AKT, Cytokine-Cytokine Recptor signal pathway and so on;4.The results of Western blot showed that the protein expression levels of P65PI3K and p53 in the single drug group and the combination group were down-regulated in varying degrees, especially in the combination group（Figure 2). Conclusion Ibrutinib combined with curcumin has synergistic killing effect on TP53- mutated CLL cells;Curcumin increases the killing effect of ibrutinib on TP53-mutated CLL cells by inhibiting PI3K and NFκB pathway, and degrading p53mt protein.

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.

Investigating the Potential Role of IKZF3 in HPV-Associated Carcinogenesis

HPVs cause approximately 5% of all human cancers. They encode two viral transforming proteins, E6 and E7, that are consistently expressed in these cancers. The HPV E6 and E7 proteins contribute to carcinogenesis by binding and functionally compromising multiple cellular regulatory proteins, most prominently the p53 and retinoblastoma (pRB) tumor suppressors, respectively. To discover additional cellular regulatory circuits that are dysregulated by E6 and E7 expression, we performed RNA sequencing analyses of HPV16 E6 and E7 expressing primary human genital epithelial cells. These experiments revealed that the Ikaros Zinc Finger 3 (IKZF3) transcription factor is highly upregulated in HPV16 E6/E7 expressing keratinocytes as compared to control keratinocytes. IKZF3 plays a key role in lymphocyte development, and when over-expressed or mutated, contributes to hematopoietic malignancies. IKZF3 is expressed at a very low level in normal epithelial cells, but the expression is upregulated in solid tumors, including invasive breast, cervical, and lung cancers. IKZF3 expression in these tumors causes upregulation of lymphocyte-specific genes, conferring lymphocyte-like behavior referred to as "lymphocyte mimicry," which presumably enables cancer cell survival in the bloodstream, thus potentially contributing to cancer metastasis. There is an FDA-approved drug, lenalidomide, that targets IKZF3 for degradation. Hence, we set out to determine whether IKZF3 might contribute to cervical carcinogenesis. We treated HPV16-positive SiHa and CaSki cervical cancer cells with 10 uM of lenalidomide for 12 hours. Cells were treated with equal amounts of DMSO as a negative control. Then, IKZF3 and E7 mRNA expression were determined using qRT-PCR after 12 hours of treatment. Gene expression was analyzed relative to GAPDH, and the results were analyzed using 2-way ANOVAs with Dunnett's correction for multiple comparisons. Western Blot experiments were performed to examine IKZF3, pRB, E7, and p53 (to indirectly assess E6) protein expression levels after 12 hours of lenalidomide treatment. Actin protein was used as a loading control. Each of these experiments were performed 3 times, qRT-PCR experiments were each performed in triplicate, and the results were graphed using a scientific 2-D graphing and statistics software. Although Western Blot showed that IKZF3 protein levels were significantly decreased by lenalidomide treatment, there was no change in pRB, E7, and p53 protein expression upon IKZF3 depletion. Similarly, there was no decrease in E7 mRNA expression. We showed by western blotting that targeting IKZF3 for proteasome-mediated degradation with lenalidomide effectively decreased the steady-state levels of IKZF3. However, we did not consistently observe an associated decrease in HPV gene expression. We are currently analyzing the transcriptome changes that IKZF3 depletion causes in cervical carcinoma cells to determine whether this may affect anoikis resistance.

Fullerene C60 protects against 7,12-dimethylbenz [a] anthracene (DMBA) induced-pancreatic damage via NF-κB and Nrf-2/HO-1 axis in rats.

The objective of this investigation was to investigate the protective effects of fullerene C60 nanoparticle against pancreatic damage experimentally induced by 7,12-dimethylbenz [a] anthracene (DMBA) in female rats. Fullerene C60 nanoparticle was administered to rats 5 times a week by oral gavage (o.g) at 1.7mg/kg bw 7days after DMBA administration. 60 Wistar albino female rats divided to four groups; Groups: (1) Control group: Fed with standard diet; (2) Fullerene C60 group: Fullerene C60 (1.7mg/kg bw); (3) DMBA group: DMBA (45mg/kg bw); (4) Fullerene C60+DMBA group: Fullerene C60 (1.7mg/kg bw) and DMBA (45mg/kg bw). Lipid peroxidation malondialdehyde (MDA), catalase activity (CAT) and glutathione (GSH) levels in pancreatic tissue were determined by spectrophotometer. Protein expression levels of p53, HO-1, p38-α (MAPK), Nrf-2, NF-κB and COX-2 in pancreatic tissue were determined by western blotting technique. In our findings, compared to the group given DMBA, MDA levels and p38-α, NF-κB and COX-2 levels decreased, CAT activity, GSH level, total protein density and p53, HO-1, Nrf-2 levels in the groups given fullerene C60 nanoparticle an increase in expression levels was observed. Our results showed that fullerene C60 nanoparticle may be more beneficial in preventing pancreatic damage.

Catalpol Ameliorates Myocardial I/R Injury Through PI3k/Akt/P53 Signaling-mediated Anti-autophagy and Anti-apoptosis

Background Myocardial infarction (MI) is a leading cardiovascular disease worldwide, with high mortality. Purpose The study aimed to observe the mechanism of catalpol inhibiting apoptosis and autophagy in H9c2 cells after ischemia/reperfusion (I/R) injury. Materials and Methods We grouped H9c2 cells into 4 groups: control, I/R injury, catalpol (I/R injury +1 ug/mL catalpol treatment), and wortmannin (I/R injury + 1 ug/mL catalpol +150 nM wortmannin treatment) groups. Both catalpol and wortmannin group cells were given drug treatment 30 min before I/R injury. At 2h post-I/R insult, we used flow cytometry for detecting cellular apoptosis and reactive oxygen species (ROS) levels. We identified p-PI3K, p-Akt, PI3K, Akt, p53, Bcl-2, Caspase-3, Bax, beclin1, LC3II, and LC3I protein expression levels. Results I/R significantly increased the apoptosis rate and ROS level of H9c2 cells, increased expression levels of p53, LC3II/LC3I, and Caspase-3, and decreased p-Akt/Akt, beclin1, Bcl-2/Bax, and p-PI3K/PI3K expression levels. Catalpol can reduce the expressions of p53, Caspase-3, and LC3II/LC3I ( p &lt; 0.5). Catalpol can increase the expression levels of Akt/ p-Akt, Bax/ Bcl-2, and beclin1. Also, it can inhibit apoptosis and autophagy levels of H9c2 cells ( p &lt; 0.5). Wortmannin, a PI3 K-specific inhibitor, was able partially to block the catalpol’s and anti-autophagic and anti-apoptotic effects. Conclusion Catalpol can inhibit apoptosis, reduces excessive autophagy, and alleviates the effects of myocardial ischemia-reperfusion through PI3K/Akt/p53 pathway.

Fructus Lycii and Salvia miltiorrhiza Bunge extract attenuate oxidative stress-induced photoreceptor ferroptosis in retinitis pigmentosa

Aim of the studyTo assess the impact of Fructus Lycii and Salvia miltiorrhiza Bunge extract (FSE) on retinitis pigmentosa (RP) and to explore the mechanisms by which FSE can prevent oxidative stress-induced photoreceptor ferroptosis in RP. MethodsHydrogen peroxide(H2O2) was used to induce oxidative stress in 661 W cells, which were then examined using flow cytometry and enzyme linked immunosorbent assay (ELISA). Changes in mitochondria were observed by using an electron microscope to characterize the ferroptosis of the cells. The protective effect of FSE on the retina function and structure of rd10 mice was evaluated using histopathological examination, fundus photographs, and electroretinography (ERG). Protein expression levels of Tumor Protein p53 (P53), Solute Carrier Family 7 Member 11 (SLC7A11), Glutathione peroxidase 4 (GPX4), Arachidonate-12-Lipoxygenase (ALOX12), and Dipeptidyl peptidase 4 (DPP4) were evaluated by Western blot assays in Vivo and in Vitro. ResultsH2O2-induced 661 W cells increased oxidative stress products and P53 and ALOX12, decreasing the expression of SLC7A11, GPX4, and DPP4. GPX4 activator does not reduce reactive oxygen species (ROS) generation and has little effect on ferroptosis. Fer-1 and FSE attenuate ROS generation and inhibit ferroptosis of photoreceptors in RP via inhibited P53 expression and increased SLC7A11 and GPX4 expression. ConclusionFSE may be available in clinical therapeutics to alleviating RP and the mechanism by which inhibits ferroptosis of photoreceptors following oxidative stress via the P53/ SLC7A11 pathway.

Pseudolaric acid B induces apoptosis associated with the mitochondrial and PI3K/AKT/mTOR pathways in triple‑negative breast cancer.

Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarixkaempferi, has been shown to exert strong antitumor properties. The aim of the present study was to investigate the mechanisms underlying the proposed antitumor properties of PAB in the triple‑negative breast cancer cells, MDA‑MB‑231. The cell processes evaluated included cell proliferation by Cell Counting Kit‑8 assay, colony formation and EdU assay, apoptosis by Annexin V‑FITC/PI apoptosis assay, cell migration by Transwell migration assay and invasion by Transwell invasion assay. PAB significantly inhibited the proliferation of MDA‑MB‑231 cells through a mechanism that was considered to be associated with cell cycle arrest at the G2/M phase. There was decreased protein expression levels of CDK1 and cyclin B1 and increased protein expression levels of p53 and p21. However, there were no well‑defined inhibitory effects on the normal breast cell line MCF10A. PAB also triggered apoptosis in a concentration‑dependent manner through the mitochondrial apoptosis pathway. It caused collapse of mitochondrial membrane potential, accumulation of reactive oxygen species and release of cytochrome c, as well as upregulation of cleaved caspase‑3, cleaved caspase‑9, cleaved PARP and Bax, and downregulation of Bcl‑2 and Bcl‑xl. The migration and invasion ability of MDA‑MB‑231 cells were inhibited by decreasing the expression levels of the epithelial‑mesenchymal transition‑related markers N‑cadherin and vimentin and increasing the expression of E‑cadherin. Moreover, the expression levels of PI3K (p110β), phosphorylated (p)‑AKT (ser473) and p‑mTOR (ser2448) were downregulated and LY294002, a PI3K inhibitor, could interact additively with PAB to induce apoptosis of MDA‑MB‑231 cells. Overall, the present results demonstrated that PAB induced apoptosis via mitochondrial apoptosis and the PI3K/AKT/mTOR pathway in triple‑negative breast cancer. It also inhibited cellular proliferation, migration and invasion, suggesting that PAB may be a useful phytomedicine for the treatment of triple‑negative breast cancer.

EXOSC10 is a novel hepatocellular carcinoma prognostic biomarker: a comprehensive bioinformatics analysis and experiment verification.

Hepatocellular carcinoma (HCC) is a common malignant tumor. There are few studies on EXOSC10 (exosome component 10) in HCC; however, the importance of EXOSC10 for HCC remains unclear. In the study, the prognosis value of EXOSC10 and the immune correlation were explored by bioinformatics. The expression of EXOSC10 was verified by tissue samples from clinical patients and in vitro experiment (liver cancer cell lines HepG2, MHCC97H and Huh-7; normal human liver cell line LO2). Immunohistochemistry (IHC) was used to detect EXOSC10 protein expression in clinical tissue from HCC. Huh-7 cells with siEXOSC10 were constructed using lipofectamine 3000. Cell counting kit 8 (CCK-8) and colony formation were used to test cell proliferation. The wound healing and transwell were used to analyze the cell migration capacity. Mitochondrial membrane potential, Hoechst 33342 dye, and flow cytometer were used to detect the change in cell apoptosis, respectively. Differential expression genes (DEGs) analysis and gene set enrichment analysis (GSEA) were used to investigate the potential mechanism of EXOSC10 and were verified by western blotting. EXOSC10 was highly expressed in tissues from patients with HCC and was an independent prognostic factor for overall survival (OS) in HCC. Increased expression of EXOSC10 was significantly related to histological grade, T stage, and pathological stage. Multivariate analysis indicated that the high expression level of EXOSC10 was correlated with poor overall survival (OS) in HCC. GO and GSEA analysis showed enrichment of the cell cycle and p53-related signaling pathway. Immune analysis showed that EXOSC10 expression was a significant positive correlation with immune infiltration in HCC. In vitro experiments, cell proliferation and migration were inhibited by the elimination of EXOSC10. Furthermore, the elimination of EXOSC10 induced cell apoptosis, suppressed PARP, N-cadherin and Bcl-2 protein expression levels, while increasing Bax, p21, p53, p-p53, and E-cadherin protein expression levels. EXOSC10 had a predictive value for the prognosis of HCC and may regulate the progression of HCC through the p53-related signaling pathway.

Differences and Similarities between Colorectal Cancer Cells and Colorectal Cancer Stem Cells: Molecular Insights and Implications.

Malignant tumors are formed by diverse groups of cancer cells. Cancer stem cells (CSCs) are a subpopulation of heterogeneous cells identified in tumors that have the ability to self-renew and differentiate. Colorectal cancer (CRC), the third most frequent malignant tumor, is progressively being supported by evidence suggesting that CSCs are crucial in cancer development. We aim to identify molecular differences between CRC cells and CRC CSCs, as well as the effects of those differences on cell behavior in terms of migration, EMT, pluripotency, morphology, cell cycle/control, and epigenetic characteristics. The HT-29 cell line (human colorectal adenocarcinoma) and HT-29 CSCs (HT-29 CD133+/CD44+ cells) were cultured for 72 h. The levels of E-cadherin, KLF4, p53, p21, p16, cyclin D2, HDAC9, and P300 protein expression were determined using immunohistochemistry staining. The migration of cells was assessed by employing the scratch assay technique. Additionally, the scanning electron microscopy method was used to examine the morphological features of the cells, and their peripheral/central elemental ratios were compared with the help of EDS. Furthermore, a Muse cell cycle kit was utilized to determine the cell cycle analysis. The HT-29 CSC group exhibited high levels of expression for E-cadherin, p53, p21, p16, cyclin D2, HDAC9, and P300, whereas KLF4 was found to be high in the HT-29. The two groups did not exhibit any statistically significant differences in the percentages of cell cycle phases. The identification of specific CSC characteristics will allow for earlier cancer detection and the development of more effective precision oncology options.

Small-molecular cyclic peptide exerts viability suppression effects on HepG2 cells via triggering p53 apoptotic pathways

Cyclic peptides have become an attractive modality for drug development due to their high specificity, metabolic stability and higher cell permeability. In an effort to explore novel antitumor compounds based on natural cyclopeptide from the phakellistatin family, we found an isoindolinone-containing analog (S-PK6) of phakellistatin 6 capable of suppressing the viability and proliferation of HepG2 cells. The aim of the present study is to shed light on the mechanism of action of this novel compound. We have detected differences in gene expression before and after treatment with S-PK6 in human hepatocellular carcinoma HepG2 cell line by transcriptome sequencing. To further investigate biological effects, we have also extensively investigated the tumor cell cycle, mitochondrial membrane potential, and intracellular Ca2+ concentration after S-PK6 treatment. Based on the finding that the apoptosis was associated with the p53 signaling pathway and MAPK signaling pathway, western blotting tests were used to assess the expression level of p53 protein and its degenerative regulator MDM2 protein, which showed that S-PK6 could increase p53 levels efficiently. In summary, our results demonstrate the mechanism of action of a small-molecule cyclopeptide, which could be very useful for examining of the possible mechanisms of natural cyclopeptides.

Formononetin Inhibits Progression of Endometriosis via Regulation of p27, pSTAT3, and Progesterone Receptor: In Vitro and In Vivo Studies.

Formononetin is one of the phytoestrogens that functions like a selective estrogen receptor modulator (SERM). In this study, we evaluated the effects of formononetin on endometriosis progression in vitro and in vivo. After pathological confirmation, 10 eutopic and ectopic endometria were collected from patients with endometriosis. Ten eutopic endometria samples were collected from patients who did not have endometriosis. To determine the cytotoxic dose and therapeutic dose of formononetin, the concentration of 70% of the cells that survived after formononetin administration was estimated using a Cell counting kit-8 (CCK 8) assay. Western blot analysis was used to determine the relative expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 in the eutopic endometria without endometriosis, eutopic endometria with endometriosis, and ectopic endometria with endometriosis as the formononetin concentration was increased. We confirmed the effect of formononetin on apoptosis and migration in endometriosis using fluorescence-activated cell sorting (FACS) and wound healing assays, respectively. A mouse model of endometriosis was prepared using a non-surgical method, as previously described. The mice were intraperitoneally administered formononetin for four weeks after dividing them into control, low-dose formononetin (40 mg/kg/day) treatment, and high-dose (80 mg/kg/day) formononetin treatment groups. All the mice were euthanized after formononetin treatment. Endometriotic lesions were retrieved and confirmed using hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) staining of p27 was performed. We set the maximum concentration of formononetin administration to 80 μM through the CCK8 assay. Based on formononetin concentration, the expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 proteins were measured using Western blot analysis (N = 4 per group). The expression level of pERK, p27, and pSTAT3 in eutopic endometrium with endometriosis tended to decrease with increasing formononetin concentration, and a significant decrease was noted at 80 μM. The expression of p27 in ectopic endometrium with endometriosis was also significantly decreased at 80 μM of formononetin. FACS analysis revealed that formononetin did not significantly affect apoptosis. In the wound healing assay, formononetin treatment revealed a more significant decrease in the proliferation of the eutopic endometrium in patients with endometriosis than in the eutopic endometrium without endometriosis. Relative expression of sex hormone receptors decreased with increasing formononetin doses. Although no significant differences were observed in the ER, PR-A, ERβ/ERα, and PR-B/PR-A, significant down-regulation of PR-B expression was noted after formononetin treatment at 80 μM. In the in vivo study, endometriotic lesions in the formononetin-treated group significantly decreased compared to those in the control group. The relative expression of p27 using IHC was highest in the control group and lowest in the high-dose formononetin treatment group. Formononetin treatment was shown to inhibit the proliferation of eutopic and ectopic endometria in patients with endometriosis through the regulation of p27, pSTAT3, and PR-B. In an endometriosis mouse model, formononetin treatment significantly reduced the number of endometriotic lesions with decreased p27 expression. The results of this study suggest that formononetin may be used as a non-hormonal treatment option for endometriosis.

Pterostilbene induces apoptosis in hepatocellular carcinoma cells: Biochemical, pathological, and molecular markers

Worldwide, hepatocellular carcinoma (HCC) is considered the sixth most prevalent cancer and ranked third in causes leading to death. Pterostilbene (PTE), a dimethylated analog of resveratrol, is a phytochemical found in fruits such as blueberries and grapes, and is known for its anticancer effect. The current study intended to investigate the effect of PTE on HepG2 cells. Cell viability, colony-forming potential, lipid peroxidation, catalase enzyme (CAT), superoxide dismutase (SOD), and caspase 3 activities, histone release, and expression levels of mTOR, S6K1, p53, and STAT3 proteins were assessed in PTE-treated HepG2 cells. In addition, the cellular and ultrastructural alterations were evaluated by light and transmission electron microscopy. PTE induced a significant reduction in HepG2 viability in a dose-dependent manner (IC50 of PTE = 74 ± 6 μM), accompanied by a decrease in colony formation potential. PTE-treated cancer cells exhibited a decrease in lipid peroxidation and CAT activity, and an increase in histone release, caspase-3, and SOD activities. Ultrastructurally, PTE-treated cells exhibited notable cell shrinkage, reduced number of filopodia, increased vacuolization, apoptotic bodies, accumulation of lipid droplets, enlarged mitochondria, dilated endoplasmic reticulum, pyknotic nuclei, and cellular fragmentation. mTOR, S6K1, and STAT3 levels were downregulated, however p53 level was modulated in PTE-treated cells. The anticancer potential of PTE might be related to its ability to alter the ultrastructure morphology, reduce mitotic activity, and modulate some key protein required for cell proliferation, suggesting its potential to trigger cancer cells towards apoptosis.

Bilirubin, an endogenous antioxidant that affects p53 protein and its downstream apoptosis/autophagy-related genes in LS180 and SW480 cell culture models of colorectal cancer

BackgroundColorectal cancer (CRC) is one of the commonest neoplasms worldwide, which its pathogenesis is strongly correlated with p53 mutations. Antioxidants are believed to decelerate the CRC progression, possibly through interfering with p53 and its downstream target genes and mechanisms. Regarding the potential antioxidant effects of bilirubin, as an incredible endogenous antioxidant, we sought to investigate how bilirubin affected the expression levels of p53 protein and its downstream target genes, including Mdm2, Bcl-2, BECN1 and LC3, in LS180 and SW480 cell culture models of CRC. Methods and resultsUsing the MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide) assay, 50 and 100 μM concentrations of bilirubin were determined to be non-toxic for both LS180 and SW480 cell lines. Western blot analysis was employed to evaluate the protein expression levels of p53. The results revealed that p53 protein levels were higher in LS180 cells treated with bilirubin compared to the control group. Notwithstanding, in SW480 cells, no considerable changes were observed in p53 protein levels of treated cells compared to the control ones. The quantitative reverse transcriptase-polymerase chain reaction (q RT-PCR) method was used to measure the mRNA expression levels of the apoptosis/autophagy-related genes, Mdm2, Bcl-2, BECN1, and LC3 , as the p53's downstream target genes. Consequently, the expression of Bcl-2 and Mdm2 genes were affected by p53, while BECN1 and LC3 expression levels were decreased in both cell lines. ConclusionBilirubin is an endogenous antioxidant with significant anti-tumor effects in the studied CRC cell lines, probably through the regulation of p53 protein expression levels and subsequent control of apoptosis and autophagy, as two key processes involved in cell survival and progression of tumor cells.

Research on Curcumin Inhibition HepG2 Human Hepatoma Cells

In recent years, with the deepening of the understanding of cancer, it has been recognized that cancer can be preventedthrough specific diets. Numerous in vitro and in vivo experimental models have also revealed that Curcumin regulatescellular signaling pathways effects of Curcumin likely activate cell death signals and induce apoptosis in cancer cells,thereby inhibiting the progression of the disease. In this study, the impact of Curcumin on the expression level of P53protein in HepG2 human hepatoma cells was observed to explore its inhibitory effect and apoptotic induction of HepG2Iand its mechanism. In this study, the MTT method will be used to detect the effect of Curcumin on the proliferationof HepG2 human liver cancer cells, and the regression calculation of Curcumin IC50 will be conducted according tothe concentration and inhibition rate. The western blot method will be used to detect the effect of ginger and flavin onthe expression of wild-type p53 in tumor cells. The effect of Curcumin on Bax apoptosis protein expression in HepG2human hepatoma cells was detected by pFT-A, a p53 inhibitor.

Effect and mechanism of Dahuang Zhechong Pills in improving liver aging in rats by regulating ROS-mediated PI3K/Akt/FoxO4 signaling pathway

Recent studies have shown that the occurrence and development of common liver diseases, including non-alcoholic fatty liver disease, cirrhosis, and liver cancer, are related to liver aging(LA). Therefore, to explore the effect and mechanism of Dahuang Zhechong Pills(DHZCP), a traditional classic prescription in improving LA with multiple targets, the present study randomly divided 24 rats into a normal group, a model group, a DHZCP group, and a vitamin E(VE) group, with six rats in each group. The LA model was induced by continuous intraperitoneal injection of D-galactose(D-gal) in rats. For the LA model rats, the general situation was evaluated by aging phenotype and body weight(BW). LA was assessed by the pathological characteristics of hepatocyte senescence, hepatic function indexes, the staining characteristics of phosphorylated histone family 2A variant(γ-H2AX), and the expression levels of cell cycle arrest proteins(P21, P53, P16) and senescence-associated secretory phenotype(SASP) in the liver. The activation of the reactive oxygen species(ROS)-mediated phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)/forkhead box protein O4(FoxO4) signaling pathway was estimated by hepatic ROS expression feature and the protein expression levels of the key signaling molecules in the PI3K/Akt/FoxO4 signaling pathway. The results showed that after the treatment with DHZCP or VE for 12 weeks, for the DHZCP and VE groups, the characterized aging phenotype, BW, pathological characteristics of hepatocyte senescence, hepatic function indexes, relative expression of ROS in the liver, protein expression levels of key signaling molecules including p-PI3K, p-Akt, and FoxO4 in the liver, staining characteristics of γ-H2AX, and the protein expression levels of P16, P21, P53, interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in the liver were improved, and the effects of DHZCP and VE were similar. Based on the D-gal-induced LA model in rats, this study demonstrates that DHZCP can ameliorate LA with multiple targets in vivo, and its effects and mechanism are related to regulating the activation of the ROS-mediated PI3K/Akt/FoxO4 signaling pathway in the liver. These findings are expected to provide new pharmacological evidence for the treatment of DHZCP in aging-related liver diseases.