G1 Phase Research Articles

LncRNA-ANRIL regulates CDKN2A to promote malignant proliferation of Kasumi-1 cells

ObjectiveThis study aimed to investigate the regulatory effects of long non-coding RNA-ANRIL on CDKN2A in the cell cycle of Kasumi-1 cells and elucidate the underlying molecular mechanisms.MethodsANRIL and CDKN2A expression levels were quantified using RT-qPCR in peripheral blood samples from acute myeloid leukemia (AML) patients. CDKN2A knockdown efficiency was validated via RT-qPCR, and cell cycle distribution was analyzed using flow cytometry. Cell proliferation assays were conducted with CCK-8 following palbociclib treatment and CDKN2A downregulation. RNA immunoprecipitation (RIP) identified potential ANRIL-associated targets, while western blotting assessed the expression levels of GSK3β/β-catenin/cyclin D1 signaling components and related proteins.ResultsANRIL and CDKN2A were markedly overexpressed in AML patient samples. Knockdown of ANRIL and CDKN2A led to G1 phase arrest accompanied by reduced CDK2/4/6 and cyclin D1 protein levels, while ANRIL upregulation induced the opposite effect. Palbociclib treatment for 24 h and 48 h elevated the G1 phase cell population and suppressed CDK2/4/6 and cyclin D1 protein expression, demonstrating its ability to counteract ANRIL-driven cell cycle progression. Downregulation of ANRIL and CDKN2A also suppressed the GSK3β/β-catenin signaling pathway, reducing cyclin D1 expression, whereas ANRIL upregulation reactivated this axis. Co-transfection experiments showed that simultaneous cyclin D1 suppression and ANRIL overexpression attenuated ANRIL’s stimulatory effects on cell cycle progression. RIP analysis confirmed a physical interaction between ANRIL and CDKN2A. Furthermore, CDKN2A downregulation inhibited cell proliferation and reversed GSK3β/β-catenin/cyclin D1 pathway activation mediated by ANRIL upregulation.ConclusionANRIL facilitates Kasumi-1 cell survival by modulating CDKN2A to activate the GSK3β/β-catenin/cyclin D1 signaling pathway.

Renal and Peripheral Blood Transcriptome Signatures That Predict Treatment Response in Proliferative Lupus Nephritis-A Prospective Study.

Mechanisms contributing to non-response to treatment in lupus nephritis (LN) are unclear. We characterised the transcriptome of paired peripheral blood mononuclear cells (PBMCs) and renal tissues in LN before and after cyclophosphamide (CYC) treatment and identified markers that predicted treatment response. Total RNA isolated from paired PBMCs (n = 32) and renal tissues (n = 25) of 16 proliferative LN before CYC treatment, 6 months post-treatment, and during renal flare, was sequenced on Illumina Novaseq-6000 platform. Post-treatment, eight patients were clinical responders (CR), of whom four flared (FL), and eight were non-responders (NR). Comparative transcriptomic analyses before and after treatment within CR, NR, and FL groups was performed using DESeq2. Weighted gene co-expression network analysis (WGCNA) and ROC analysis was performed to identify and validate hub genes predictive of treatment response. Based on this, we observed that pathways such as degradation of cell cycle proteins, expression of G0 and G1 phase proteins, and apoptosis, were upregulated in CR PBMCs post-treatment, while IFN-γ signalling and ECM organisation were downregulated. In NR PBMCs, ECM molecules, neddylation and BCR signalling were upregulated post-CYC treatment, while in NR renal tissue, TLR, IFN and NF-κB signalling pathways were upregulated. In FL PBMCs, neutrophil degranulation and ROS and RNS production in phagocytes were downregulated following treatment, whereas, in the corresponding renal tissue, cell-ECM interactions and ISG15 antiviral mechanism were downregulated. After WGCNA and subsequent ROC analysis, TENM2, NLGN1 and AP005230.1 from PBMCs each predicted NR (AUC-0.91; p = 0.03), while combined model improved prediction (AUC-0.94; p = 0.02). AP005230.1 from renal tissue also predicted non-response (AUC-0.94; p = 0.01) and AC092436.3 from PBMCs predicted renal flare (AUC-0.81; p = 0.04). Our study identified significant DEGs/pathways specific to different treatment outcomes and hub genes that predicted non-response and renal flare.

The Role of WDR77 in Cancer: More Than a PRMT5 Interactor.

WD repeat domain 77 protein (WDR77), a WD-40 domain-containing protein, is a crucial regulator of cellular pathways in cancer progression. While much of the past research on WDR77 has focused on its interaction with PRMT5 in histone methylation, WDR77's regulatory functions extend beyond this pathway, influencing diverse mechanisms such as mRNA translation, chromatin assembly, cell cycle regulation, and apoptosis. WDR77 is a key regulator of cell cycle progression, regulating the transition from the G1 phase. WDR77 regulates many signaling pathways such as TGFβ where its role in these cellular pathways underscores its broad oncogenic potential. WDR77 also assists and promotes certain transcription factors such as E2F. Furthermore, in certain cancers, WDR77 enhances steroid hormone receptor activity, uniquely linking it to hormone-driven malignancies. WDR77 often translocates between the nucleus and the cytoplasm, with its location dictating its role in the cell. WDR77 has the ability to adapt its function depending on its location which emphasizes its dynamic role in both promoting and inhibiting tumor growth, depending on cellular context. This dual function makes WDR77 an attractive therapeutic target, as disrupting its interactions with critical signaling pathways or modulating its translocation could yield novel strategies for cancer treatment. Given WDR77's role in oncogenic pathways independent of PRMT5, further exploration of WDR77 and its non-PRMT5-related activities may reveal additional therapeutic opportunities in an array of cancers.

Gintonin-Enriched Panax ginseng Extract Induces Apoptosis in Human Melanoma Cells by Causing Cell Cycle Arrest and Activating Caspases

Gintonin, a non-saponin glycolipoprotein from Panax ginseng, acts as a lysophosphatidic acid ligand. However, its anticancer effects, especially in melanoma, remain unclear. This study investigated the anti-proliferative effects and intracellular signaling mechanisms of a gintonin-enriched fraction (GEF) from Panax ginseng in human melanoma cell lines. In vitro, GEF treatment significantly inhibited cell proliferation, reduced clonogenic potential, and delayed wound healing in melanoma cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining showed that GEF induced apoptosis, as evidenced by increased apoptotic cell populations and nuclear changes. GEF also caused cell cycle arrest in the G1 phase for A375 cells and the G2/M phase for A2058 cells. It triggered apoptotic signaling via activation of caspase-3, -9, poly (ADP-ribose) polymerase cleavage, and downregulation of B cell lymphoma-2 (Bcl-2). GEF treatment also raised intracellular reactive oxygen species (ROS) levels and mitochondrial stress, which were mitigated by N-acetyl cysteine (NAC), an ROS inhibitor. In vivo, GEF suppressed tumor growth in A375- and A2058-xenografted mice without toxicity. These findings suggest that GEF from Panax ginseng has potential antitumor effects in melanoma by inducing apoptosis and cell cycle arrest, presenting a promising therapeutic avenue.

Synthesis, Anticancer Screening, and In Silico Evaluations of Thieno[2,3-c]pyridine Derivatives as Hsp90 Inhibitors

Background: Thieno[2,3-c]pyridines and their analogs are not well explored for their anticancer properties. Hence, our research aimed to establish the anticancer potential of thieno[2,3-c]pyridines through cell-based assays and in silico evaluations. Methods: Thieno[2,3-c]pyridine derivatives 6(a–k) were synthesized and characterized using FT-IR, 1H-NMR, 13C-NMR, and HRMS. All the synthesized compounds were screened initially for their anticancer activity against MCF7 and T47D (breast cancer), HSC3 (head and neck cancer), and RKO (colorectal cancer) cell lines using MTT assay. Apoptosis and cell cycle analyses were conducted using Annexin V/propidium iodide (PI) double staining for apoptosis assessment and PI staining for cell cycle analysis to investigate the mechanisms underlying the reduced cell viability. In silico molecular docking was accomplished for the synthesized compounds against the Hsp90 and determined pharmacokinetics properties. Results: From the screening assay, compounds 6a and 6i were identified as potential inhibitors and were further subjected to IC50 determination. The compound 6i showed potent inhibition against HSC3 (IC50 = 10.8 µM), T47D (IC50 = 11.7 µM), and RKO (IC50 = 12.4 µM) cell lines, all of which indicated a broad spectrum of anticancer activity. Notably, 6i was found to induce G2 phase arrest, thereby inhibiting cell cycle progression. Molecular docking results indicated crucial molecular interactions of the synthesized ligands against the target Hsp90. Conclusion: The compound 6i induced cell death via mechanisms that are different from apoptosis. Thus, the synthesized thieno[2,3-c]pyridine derivatives can be suitable lead compounds to be optimized to obtain potent anticancer agents through Hsp90 inhibition.

Expression, characterization and anti-colon cancer activity of recombinant ginseng peptides with amino acid tandem repeats.

Ginseng peptides, small molecule active ingredients in ginseng, are mainly extracted naturally or synthesised chemically, but high costs and difficulties hinder further research. In this study, a ginseng hexapeptide FKEHGY, named antitumor peptide 0601 (AT0601) and its five tandem sequence repeats AT0605, were expressed in Bacillus subtilis WB600 for the first time, and the bioactivity study showed that the anticancer activity of AT0605 was even significantly higher than that of AT0601 for colon cancer CT26 cells, with IC50s of 16.82±1.3 μM (48 h) and 855.57±6.04 μM (48 h), respectively, i.e. AT0605 achieves the same inhibition rate for CT26 cells at a concentration 50 times lower than that of AT0601. Similar to the ginseng peptide AT0601, recombinant AT0605 also inhibited cell growth by blocking cells in the G1 phase and activated the mitochondria-associated caspase pathway to induce apoptosis. In conclusion, all two kinds of the recombinant ginseng peptides could also inhibit cell proliferation through mechanisms such as inhibiting cell cycle arrest and inducing a decrease in mitochondrial membrane potential.

Calothrixin B by docking JAK2 is a potential therapeutic inhibitor for pancreatic ductal adenocarcinoma.

Pancreatic cancer, a highly invasive and prognostically unfavorable malignant tumor, consistently exhibits resistance to conventional chemotherapy, leading to substantial side effects and diminished patient quality of life. This highlights the critical need for the discovery of novel, effective, and safe chemotherapy drugs. This study aimed to explore bioactive compounds, particularly natural products, as an alternative for JAK2 protein inhibitor in cancer treatment. Molecular docking, molecular dynamics, and Western blot experiments were conducted to verify the binding of Calothrixin B to JAK2 and its inhibitory effect on the JAK2-STAT3 signaling axis. Recognizing the significant impact of JAK-STAT3 signaling pathway in pancreatic cancer, we screened the Zinc database to discover potential JAK2 inhibitors, and identified the small molecule Calothrixin B as a promising drug. Molecular simulations revealed stable interactions and the formation of hydrogen bonds between Calothrixin B and specific amino acids (Asp 994, Leu 855, and Arg 980) after a 100 ns simulation. Furthermore, we show that Calothrixin B inhibited the activity of the JAK2-STAT3 signaling pathway, arrested pancreatic cancer cells in the G1 phase, induced apoptosis, and significantly inhibited cell migration. Moreover, in vivo on a subcutaneous tumor model in nude mice confirmed that Calothrixin B effectively inhibited tumor growth in nude mice. In addition, the combination of Carlothrixin B and gemcitabine had a better inhibitory effect on pancreatic cancer cells. These findings introduce new avenues for Calothrixin B as promising therapy for pancreatic cancer.

Identification of KRT16 and ANXA10 as cell cycle regulation genes for lung adenocarcinoma based on self-transcriptome sequencing of surgical samples and TCGA public data mining

AimThis study aimed to identify the genes associated with the development of lung adenocarcinoma (LUAD) and potential therapeutic targets.MethodsDifferentially expressed genes (DEGs) were identified by self-transcriptome sequencing of tumor tissues and paracancerous tissues resected during surgery and combined with The Cancer Genome Atlas (TCGA) data to screen for the genes associated with LUAD prognosis. The expression was validated at mRNA and protein levels, and the gene knockdown was used to examine the impact and underlying mechanisms on lung cancer cells.ResultsA total of 227 DEGs were identified by transcriptome sequencing, and the 20 DEGs with the most significant differences were used for co-analysis with TCGA data. The findings suggested that KRT16 and ANXA10 might have an important role in the development of LUAD after validating the mRNA and protein expression levels at the cellular level. The knockdown of KRT16 and ANXA10 inhibited the proliferation of lung cancer cells, and the cell cycle was blocked in the G1 phase. The expression of the G1/S–phase cell cycle checkpoint–related proteins cyclin D1 and cyclin E was inhibited by KRT16 and ANXA10 knockdown, respectively. The tumor formation ability decreased after KRT16 or ANXA10 knockdown in vivo.ConclusionsKRT16 and ANXA10 are potential genes regulating the development of LUAD. Also, they may be potential targets for the targeted therapy of LUAD by inhibiting the proliferation of lung cancer cells and blocking the cell cycle by affecting key protein expression levels at cell cycle checkpoints.

Glut-1 exerts anti-tumor activity in glioma by regulating AKT / mTOR signaling pathway

Objective Glioma is a common tumor in neurosurgery. Glut-1 is the main carrier of glucose uptake by cells and can provide energy through the glycolytic pathway. However, the role and related mechanisms of Glut-1 in glioma have not yet been elucidated. Methods Real time PCR and Western blot were done to assess Glut-1 level in glioma tumor tissues and adjacent tissues. Glioma U87 cells were separated into control group; Glut-1 negative control (NC group); and Glut-1 siRNA group followed by analysis of Glut-1 expression, cell proliferation by MTT assay, cell invasion, cell apoptosis and cycle by flow cytometry and AKT / mTOR signaling proteins level by Western blot. Results Glut-1 expression was significantly increased in the tissues of glioma patients (P < 0.05) compared to adjacent tissues and its level was related to tumor size, pathological grade and survival. Down-regulating the expression of Glut-1 can significantly inhibit tumor cell proliferation and invasion, increase apoptosis and induce G0 phase of cell cycle arrest, and inhibit the expression of AKT / mTOR signaling proteins phosphorylation (P < 0.05). Conclusions Glut-1 level in glioma tissues is significantly increased, which is related to the pathological features. Down-regulating Glut-1 can inhibit glioma by regulating the AKT / mTOR signaling pathway.

New molecular hybrids integrated with quinoxaline and pyrazole structural motifs: VGFR2 inhibitors and apoptosis inducers.

The vascular endothelial growth factor receptor is essential for the angiogenesis of cancer. Tumor propagation was effectively suppressed by inhibiting VEGFR-2 activity. As a result, the target quinoxaline-pyrazole hybrids were created in a way that closely resembled the structural characteristics of VEGFR-2 inhibitors. The synthesized compounds were assessed in vitro using the MTT assay with doxorubicin serving as a reference standard for their cytotoxic properties against the HCT-116 and MCF-7 cell lines. Additionally, when tested on human normal fibroblasts (WI38), the promising cytotoxic compounds 8, 11, 13, and 15 were shown to be selective to cancer cells. Using ELISA, they showed mechanistically inhibitory activities against VEGFR-2; compound 13 was the most effective inhibitor, with an IC50 of 0.045±6.24 uM, surpassing sorafenib (IC50 of 0.049±5.24 μM). Notably, it was discovered that our target compound, 13, was 1.1 times more potent than sorafenib and 3.19 times more potent than sunitinib as a VGFRA2 inhibitor. Furthermore, Western blot analysis revealed that its VEGFR2 protein levels were noticeably higher than the control. Compound 13's selectivity towards VEGFR2 was further confirmed by testing it against other kinases, such as PDGFRA (IC50 0.329±0.014 μM) and EGFR (IC50 0.6±0.019 μM). Furthermore, 13 demonstrated a 50% decrease in VEGF-A secretion in comparison to the control group, demonstrating its anti-angiogenic quality. A scratch closure percentage of 57.78%, which was much lower than the 97.04 percent of untreated control cells, showed 13's anti-migratory capability. According to the cell cycle study, compound 13 induces apoptosis at the sub-G1 phase and terminates the cell cycle at the G1 phase. Consequently,flow cytometric analysis revealed that it caused apoptosis; compound 13 increases the BAX/Bcl-2 ratio from control to 13.66, and it also activates caspase 3 to 422.48±43.82 and induces p53 to 366.79±40.21. Docking simulations revealed potential binding modes and crucial structural elements of active drugs, and they were almost in agreement with enzymatic examination. For every hybrid, in silico physicochemical attributes, drug likeness metrics, and ligand efficiency were plausible. It's interesting to note that 13 and 15 are plausible medication candidates.

The Role of NF-κB/MIR155HG in Regulating the Stemness and Radioresistance in Breast Cancer Stem Cells.

Breast cancer stem cells (BCSCs) are instrumental in treatment resistance, recurrence, and metastasis. The development of breast cancer and radiation sensitivity is intimately pertinent to long non-coding RNA (lncRNA). This work is formulated to investigate how the lncRNA MIR155HG affects the stemness and radioresistance of BCSCs. Effects of MIR155HG knockdown on BCSCs were gauged in MCF-7 and MDA-MB-231 cell lines. MIR155HG expression was manipulated in cells, followed by an assessment of stemness, DNA damage repair, apoptosis, cell cycle, and the Wnt signaling pathway under radiation conditions. The interaction between nuclear factor kappa B (NF-κB) subunit RelA and MIR155HG was examined using a dual-luciferase reporter assay. To examine the binding interaction between RelA and MIR155HG promoter, chromatin immunoprecipitation was performed. Breast cancer-derived stem cells exhibited a high level of MIR155HG. Knockdown of MIR155HG reduced stemness, enhanced radiosensitivity, induced apoptosis, and arrested cells in the G1 phase. Mechanistically, MIR155HG knockdown repressed Wnt/β-catenin signaling and mediated apoptosis-related protein expressions. NF-κB subunit RelA transcriptionally activated MIR155HG, thereby contributing to radioresistance in BCSCs. NF-κB regulates MIR155HG transcriptionally to activate the Wnt pathway, thus enhancing stemness and radioresistance in BCSCs. Targeting MIR155HG may enhance the susceptibility of cancer stem cells to radiation-induced cell death, potentially improving therapeutic outcomes. These findings underscore MIR155HG as a promising therapeutic target for breast cancer.

Methyltransferase-like 7B participates in bladder cancer via ACSL3 m6A modification in a ferroptosis manner

BackgroundBladder cancer (BC) is a malignant tumor. Methyltransferase-like 7B (MEETL7B) is a methyltransferase and its role in BC has not yet been revealed.MethodStable METTL7B knockdown or overexpression were achieved by lentiviral transduction in SW780 and TCCSUP cell lines. Xenografts tumors were established via subcutaneous injection of stable transfectants in BALB/c mice.ResultsA database search indicated that METTL7B was elevated in BC and it was validated in BC cell lines. METTL7B silencing suppressed cell proliferation and tumorigenesis in vitro and in vivo. Besides, METTL7B knockdown induced cell cycle arrest in G1 phase with a reduction in cyclin D1(CCND1), CDK4, and CDK6 levels and an elevation in CDKN2D levels in cells. Considering that ferroptosis is emerging as a therapeutic target for cancer, and the possible relationship between METTL7B and antioxidant enzymes. We, here, examined that ectopic METTL7B expression abolished ferroptosis markers in cells raised by Erastin treatment, including the production of lipid ROS, the increased cellular iron and MDA content, the decreased gene expression of ACSL3, FANCD2, and FADS2, as well as the mitochondrial injury observed by electron microscopy. Mechanically, ectopic METTL7B expression promoted m6A modification on ACSL3 mRNA. Gain of functional experiment exhibited that METTL7B inhibited Erastin-induced ferroptosis via ACSL3. Overexpressed PLAGL2 is identified as a possible independent predictor in BC and bioinformatics predicted the potential binding sites between PLAGL2 and METTL7B promoter region. Dual luciferase and chromatin immunoprecipitation analysis provided evidence that PLAGL2 directly binds to METTL7B promoter region.ConclusionsMETTL7B is involved in BC development and progression. METTL7B may mediate m6A modification on ACSL3 mRNA to negatively regulate ferroptosis in BC cells, which provides a potential therapeutic target for BC via ferroptosis.Graphical

Inhibiting miR-155-5p promotes proliferation of human submandibular gland epithelial cells in primary Sjogren's syndrome by negatively regulating the PI3K/AKT signaling pathway via PIK3R1.

To investigate the mechanism mediating the regulatory effect of miR-155-5p on proliferation of human submandibular gland epithelial cells (HSGECs) in primary Sjogren's syndrome (pSS). Dual luciferase reporter assay was used to verify the targeting relationship between miR-155-5p and the PI3K/AKT pathway. In a HSGEC model of pSS induced by simulation with TRAIL and INF-γ, the effects of miR-155-inhibitor-NC or miR-155 inhibitor on cell viability, cell cycle, apoptosis and proliferation were evaluated using CKK8 assay, flow cytometry and colony formation assay. ELISA and RT-PCR were used to detect the expressions of inflammatory cytokines and miR-155-5p mRNA in the cells; Western blotting was performed to detect the expressions of proteins in the PI3K/AKT signaling pathway. Dual luciferase assay showed that miR-155-5p targets the PI3K/AKT pathway via PIK3R1 mRNA. The HSGEC model of pSS showed significantly decreased cell viability, cell clone formation ability and expressions IL-10 and IL-4 and increased cell apoptosis, cell percentage in G2 phase, expressions of TNF‑α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-Akt/AKT ratio, and PIK3R1 protein expression. Treatment of the cell models with miR-155 inhibitor significantly increased the cell viability, G1 phase cell percentage, colony formation ability, and expressions of IL-10 and IL-4 levels, and obviously reduced cell apoptosis rate, G2 phase cell percentage, expressions of TNF-α, IL-6, miR-155-5p and PIK3R1 mRNA, p-PI3K/PI3K ratio, p-AKT/AKT ratio, and PIK3R1 protein expression. In HSGEC model of pSS, inhibition of miR-155-5p can promote cell proliferation and reduced cell apoptosis by targeting PI3K1 mRNA to negatively regulate the overexpression of PI3K/AKT signaling pathway.

Design, Synthesis, Anticancer Screening, and Mechanistic Study of Spiro-N-(4-sulfamoyl-phenyl)-1,3,4-thiadiazole-2-carboxamide Derivatives.

The present study aims to create spiro-N-(4-sulfamoyl-phenyl)-1,3,4-thiadiazole-2-carboxamide derivatives with anticancer activities. The in vitro anticancer evaluation showed that only the novel spiro-acenaphthylene tethered-[1,3,4]-thiadiazole (compound 1) exhibited significant anticancer efficacy as a selective inhibitor of tumor-associated isoforms of carbonic anhydrase. Compound 1 demonstrated considerable efficacy against the renal RXF393, colon HT29, and melanoma LOX IMVI cancer cell lines, with IC50 values of 7.01 ± 0.39, 24.3 ± 1.29, and 9.55 ± 0.51 µM, respectively. In comparison, doxorubicin exhibited IC50 values of 13.54 ± 0.82, 13.50 ± 0.71, and 6.08 ± 0.32 µM for the corresponding cell lines. Importantly, compound 1 exhibited lower toxicity to the normal WI 38 cell line than doxorubicin, with IC50 values of 46.20 ± 2.59 and 18.13 ± 0.93 µM, respectively, indicating greater selectivity of the target compound compared to the standard anticancer agent doxorubicin. Also, mechanistic experiments demonstrated that compound 1 exhibits inhibitory activity against human carbonic anhydrase hCA IX and XII, with IC50 values of 0.477 ± 0.03 and 1.933 ± 0.11 μM, respectively, indicating enhanced selectivity for cancer-associated isoforms over cytosolic isoforms hCA I and II, with IC50 values of 7.353 ± 0.36 and 12.560 ± 0.74 μM, respectively. Cell cycle studies revealed that compound 1 caused G1 phase arrest in RXF393 cells, and apoptosis experiments verified a substantial induction of apoptosis with significant levels of early and late apoptosis, as well as necrosis (11.69%, 19.78%, and 3.66%, respectively), comparable to those induced by the conventional cytotoxic agent doxorubicin, at 9.91%, 23.37%, and 6.16%, respectively. Molecular docking experiments confirmed the strong binding affinity of compound 1 to the active sites of hCA IX and XII, highlighting significant interactions with zinc-binding groups and hydrophobic residues. These findings underscore the target compound's potential as a viable anticancer agent via targeting CA.

Regeneration of Vascular Endothelium in Different Large Vessels.

The regeneration of endothelial cells (ECs) lining arteries, veins, and large lymphatic vessels plays an important role in vascular pathology. To understand the mechanisms of atherogenesis, it is important to determine what happens during endothelial regeneration. A comparison of these processes in the above-mentioned vessels reveals both similarities and some significant differences. Regeneration is carried out by moving intact ECs from the edges of the viable endothelial layer towards the centre of the EC damage zone. A sharp decrease in contact inhibition leads to the spreading of the edges of the ECs situated on the damage border. This stimulates the second row of ECs to enter the S-phase, then the G2 phase of cell cycle, and finally mitosis. In all three types of vessels studied, mitotically dividing ECs were found using correlation light and electron microscopy. These ECs have a body protruding into the lumen of the vessel, covered with micro-villi and other outgrowths. The level of EC rounding and protruding is highest in the arteries and least pronounced in the lymphatic vessels. The intercellular contacts of mitotically dividing cells become wider. The EC division leads to an increase in the density of ECs. ECs moving over the damaged area and partially outside the damaged area acquire a fusiform shape. In the process of regeneration of arterial endothelium, the damaged ECs are removed. Then health ECs move to a surface devoid of endothelium, and detach spreading out, flattened platelets from the luminal surface of the vessel. In the veins, ECs grow on the surface of platelets and microthrombi. In lymphatic vessels, ECs detach from the basement membrane slower than in the veins and arteries. There, the migrating ECs grow under fibrin fibres. After some time (usually after 30 days), the EC mosaic returns to normal in all three types of vessels.

In vitro antitumor effects of methanolic extracts of three Ganoderema mushrooms

Ganoderma mushrooms have a variety of pharmacological activities and may have antitumor effects. Therefore, the antitumor activity of the methanolic fruiting body extracts of three Ganoderma spp. will be evaluated by estimating cell viability, cell cycle parameters and the mode of cellular death. In this regard, Sulfo-rhodamine B staining and flow cytometry were used. Hepatocellular carcinoma (HepG2) and breast ductal carcinoma (T-47D) cell lines were used as cancer models, while mouse normal liver (BNL) and oral epithelial cell (OEC) lines were used as respective controls. The results revealed that Ganoderma resinaceum extract decreased the viability of BNL at an IC50 > 100 µg/mL but not that of HepG2 at an IC50 of 72.32 µg/mL. Additionally, Ganoderma australe and Ganoderma mbrekobenum decreased the viability of OEC cell line at an IC50 of 328.29 and 271.56 µg/ mL, respectively. On the other hand, the IC50 of T-47D were 221.95 and 236.45 µg/mL, respectively. The three extracts arrested the cell life cycle at the G1 phase in each case. G. resinaceum extract stimulated total apoptosis (Q2 + Q4) of 19.99% with low necrosis (Q1). However, the percentages of total cell necrosis in the T-47D cell line treated with the other two extracts were 31.10% and 18.28%, respectively while the percentages of total cell apoptosis were 6.83% and 1.78%, respectively. Thus, G. resinaceum significantly inhibited the viability of the HepG2 cell line, while both the G. australe and G. mbrekobenum extracts significantly decreased the viability of the T-47D cell line. These results may encourage speculation about their possible use for the therapeutic management of hepatocellular carcinoma and breast ductal carcinoma after further investigation.

Dual regulation of the levels and function of Start transcriptional repressors drives G1 arrest in response to cell wall stress

BackgroundMany different stress signaling pathways converge in a common response: slowdown or arrest cell cycle in the G1 phase. The G1/S transition (called Start in budding yeast) is a key checkpoint controlled by positive and negative regulators. Among them, Whi7 and Whi5 are transcriptional repressors of the G1/S transcriptional program, yeast functional homologs of the Retinoblastoma family proteins in mammalian cells. Under standard conditions, Whi7 plays a lesser role than Whi5 in Start inhibition. However, under cell wall stress, Whi7 is induced and plays a more important role in G1/S control. In this work, we investigated the functional hallmarks of Whi7 and Whi5, which determine their strength as Start inhibitors under cell wall stress.MethodsThe response of Saccharomyces cerevisiae to Calcofluor White was investigated to characterize the regulation and function of Whi7 and Whi5 under cell wall stress. To control their protein levels, we used dose-dependent β-estradiol-induced expression and auxin-induced degron protein fusions. We also performed Chromatin Immunoprecipitation assays to investigate Whi7 and Whi5 association with Start promoters and scored cell cycle arrest and re-entry using cell microscopy assays.ResultsWe found that cell wall stress promoted the specific upregulation of the Whi7 Start repressor. First, although cell wall stress increases Whi7 protein levels, this is not the only determinant behind the Whi7 function in promoting G1 arrest. Indeed, artificial induction of Whi5 at the same protein level resulted in a lower G1 block. Second, under cell wall stress, Whi7 was specifically recruited to SBF-target promoters, independent of the increase in its protein levels or cell cycle stage. Finally, we found that Whi7 protein instability further increased during cell wall stress and that Whi7 degradation triggered advanced cell cycle re-entry.ConclusionsHere, we show that cell wall stress signaling specifically enhances Whi7 function as a Start transcriptional repressor. Importantly, we identified new Whi7-specific regulatory mechanisms that do not operate in the Whi5 repressor. Our results indicate that cells may benefit from stress-specific repressors to ensure the stress-induced G1 arrest and that Whi7 rapid degradation may be particularly important to resume cell cycle upon adaptation.

Single-cell and bulk RNA sequencing analysis reveals CENPA as a potential biomarker and therapeutic target in cancers.

Cancer remains one of the most significant public health challenges worldwide. A widely recognized hallmark of cancer is the ability to sustain proliferative signaling, which is closely tied to various cell cycle processes. Centromere Protein A (CENPA), a variant of the standard histone H3, is crucial for selective chromosome segregation during the cell cycle. Despite its importance, a comprehensive pan-cancer bioinformatic analysis of CENPA has not yet been conducted. Data on genomes, transcriptomes, and clinical information were retrieved from publicly accessible databases. We analyzed CENPA's genetic alterations, mRNA expression, functional enrichment, association with stemness, mutations, expression across cell populations and cellular locations, link to the cell cycle, impact on survival, and its relationship with the immune microenvironment. Additionally, a prognostic model for glioma patients was developed to demonstrate CENPA's potential as a biomarker. Furthermore, drugs targeting CENPA in cancer cells were identified and predicted using drug sensitivity correlations and protein-ligand docking. CENPA exhibited low levels of gene mutation across various cancers. It was found to be overexpressed in nearly all cancer types analyzed in TCGA, relative to normal controls, and was predominantly located in the nucleus of malignant cells. CENPA showed a strong association with the cancer cell cycle, particularly as a biomarker for the G2 phase. It also emerged as a valuable diagnostic and prognostic biomarker across multiple cancer types. In glioma, CENPA demonstrated reliable prognostic potential when used alongside other prognostic factors. Additionally, CENPA was linked to the immune microenvironment. Drugs such as CD-437, 3-Cl-AHPC, Trametinib, BI-2536, and GSK461364 were predicted to target CENPA in cancer cells. CENPA serves as a crucial biomarker for the cell cycle in cancers, offering both diagnostic and prognostic value.

MYC Overexpression Enhances Sensitivity to MEK Inhibition in Head and Neck Squamous Cell Carcinoma.

MEK inhibitors, such as trametinib, have shown therapeutic potential in head and neck squamous cell carcinoma (HNSCC). However, the factors influencing cancer cell sensitivity and resistance to MEK inhibition remain poorly understood. In our study, we observed that MEK inhibition significantly reduced the expression of MYC, a transcription factor critical for the therapeutic response. MYC overexpression markedly enhanced the sensitivity of HNSCC cells to trametinib, as evidenced by delayed wound healing and reduced colony formation. Cell cycle analysis revealed that trametinib induced a G1 phase arrest, whereas MYC overexpression accelerated cell cycle progression, with a reduced induction of p27 and p21 and diminished decreases in E2F1 and phospho-Ser2/5 levels. Flow cytometry and protein analyses demonstrated that MYC overexpression amplified trametinib-induced apoptosis and DNA damage, as evidenced by elevated levels of pro-apoptotic markers (p53, cleaved PARP, and BIM) and γH2AX. In vivo xenograft models confirmed these findings, showing increased sensitivity to trametinib in MYC-overexpressing tumors. Moreover, MEK inhibition increased autophagy in HNSCC cells, a factor critical for therapeutic resistance. Inhibiting trametinib-induced autophagy further enhanced apoptotic cell death. These findings suggest that MYC expression and autophagy play crucial roles in HNSCC's response to MEK inhibition. Combining trametinib with autophagy inhibition may improve therapeutic outcomes in HNSCC.

Design and synthesis of new 1,2,3-triazole derivatives as VEGFR-2/telomerase downregulatory candidates endowed with apoptotic potential for cancer treatment.

In this current work, we dedicated efforts to designing and synthesizing new 1,2,3-triazole-analogues (5a-d), (6a-d), and (7a-c) to act as dual VEGFR-2 and telomerase inhibitors with promising apoptotic potential. The synthesized analogues were examined against eleven diverse types of cancer cells and two normal cells to assess their ability to inhibit cell growth (GI%). Obviously, compound 7b showed the best average GI% (75.69%) surpassing the average GI% of Dox (65.79%). Compound 5d showed the lowest IC50 values (25.86 and 51.91µM) against HNO-97 and FaDu cancer cells, respectively. Besides, compound 5a exhibited the lowest IC50 value (15.46µM) against HCT116, whereas compound 6b revealed the lowest IC50 value (31.14µM) against HuH7. Besides, candidates 5a, 5b, 5d, and 7a showed prominent inhibitory results towards VEGFR-2 protein with decreasing its expression by 0.33, 0.42, 0.38 and 0.26-fold change, respectively. However, compounds 5a, 5b, 5d, and 7a showed promising inhibitory results towards telomerase protein and decreased its expression by 0.60, 0.50, 0.52, and 0.44-fold change, respectively. Additionally, it was clear that compound 5a was able to upregulate the expression of Caspases 3, 8, and 9 proteins by 2.19, 1.83, and 1.62-fold change, respectively. Besides, 5a was able to downregulate the expression of CDK-2, CDK-4, and CDK-6 proteins by 0.50, 0.43, and 0.13-fold change, respectively. Obviously, compound 5a halted the cell cycle at the G1, S, and G2-M phases in HCT116 cells. Subsequently, the synthesized 1,2,3-triazole analogues can be treated as lead VEGFR-2 and telomerase inhibitors with potential apoptotic activity for future optimization and cancer treatment.