Expression of Cyclin D1 in Urothelial Carcinoma of Urinary Bladder and its Association with Tumour Grade.

Urothelial carcinoma (UC) is the most common malignancy of urinary bladder. It is the 9th leading cause of death worldwide and second most common genitourinary malignancy among male. Incidence is increasing in developing countries like Bangladesh. About 80% of patients are found between 50 to 80 years of age. It is 3-4 times more common in male than in female. Determination of therapeutic strategy and prediction of progression of urothelial carcinoma is a major clinical challenge. Treatment of urothelial carcinoma still now mostly depends on pathological stages. Amplification or genomic alteration of Cyclin D1 (a proto-oncogene) may cause protein overexpression which is frequently realized as a clonal pathology in various human neoplasms including bladder cancer. Evaluation of Cyclin D1 expression is promising for guiding therapeutic strategies, risk stratification and prediction of tumor progression. The aim of the study was to determine the expression of Cyclin D1 in urothelial carcinoma of urinary bladder and its association with tumour grade. This cross-sectional observational study was conducted in Department of Pathology, Dhaka Medical College, Dhaka, Bangladesh from July 2019 to June 2021. Histomorphologically diagnosed 51 urothelial carcinomas were included. Sections were stained with hematoxylin and eosin. Immunostaining with Cyclin D1 antibody was also done. Relevant information was collected and recorded in a predesigned data sheet. Statistical analysis was carried out as required. Mean age ±SD was 57.8±10.55 years. Male female ratio was 4.6:1. In this study 39(76.5%) patients were smoker. Regarding clinical presentations 36(70.6%) patients presented with painless hematuria alone. Lateral wall (64.7%) was the most frequent tumor location. Among 51 cases, 38(74.5%) cases were high grade urothelial carcinoma (HGUC) and 13(25.5%) cases were low grade urothelial carcinoma (LGUC). Considering Cyclin D1 expression, most of the LGUC cases showed high level of expression by both percentage (84.6%) and intensity (84.6%). Most of the HGUC cases showed low level of expression by both percentage (63.2%) and intensity (60.5%). Cyclin D1 showed significant inverse association with HGUC (p<0.05). In urothelial carcinoma of urinary bladder, Cyclin D1 expression was decreased with increasing grade of the tumor. Cyclin D1 expression was inversely associated with tumour grade.

[Banxia Xiexin Decoction inhibiting colitis-associated colorectal cancer infected with Fusobacterium nucleatum by regulating Wnt/ß-catenin pathway].

This paper investigates the intervention effect and mechanism of Banxia Xiexin Decoction(BXD) on colitis-associated colorectal cancer(CAC) infected with Fusobacterium nucleatum(Fn). C57BL/6 mice were randomly divided into a control group, Fn group, CAC group [azoxymethane(AOM)/dextran sulfate sodium salt(DSS)](AOM/DSS), model group, and BXD group. Except for the control and AOM/DSS groups, the mice in the other groups were orally administered with Fn suspension twice a week. The AOM/DSS group, model group, and BXD group were also injected with a single dose of 10 mg·kg~(-1) AOM combined with three cycles of 2.5% DSS taken intragastrically. The BXD group received oral administration of BXD starting from the second cycle until the end of the experiment. The general condition and weight changes of the mice were monitored during the experiment, and the disease activity index(DAI) was calculated. At the end of the experiment, the colon length and weight of the mice in each group were compared. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in the colon tissue. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of interleukin(IL)-2, IL-4, and IL-6 inflammatory factors in the serum. Immunohistochemistry(IHC) was used to detect the expression of Ki67, E-cadherin, and ß-catenin in the colon tissue. Western blot was used to detect the protein content of Wnt3a, ß-catenin, E-cadherin, annexin A1, cyclin D1, and glycogen synthase kinase-3ß(GSK-3ß) in the colon tissue. The results showed that compared with the control group, the Fn group had no significant lesions. The mice in the AOM/DSS group and model group had decreased body weight, increased DAI scores, significantly increased colon weight, and significantly shortened colon length, with more significant lesions in the model group. At the same time, the colon histology of the model group showed more severe adenomas, inflammatory infiltration, and cellular dysplasia. The levels of IL-4 and IL-6 in the serum were significantly increased, while the IL-2 content was significantly decreased. The IHC results showed low expression of E-cadherin and high expression of Ki67 and ß-catenin in the model group, with a decreased protein content of E-cadherin and GSK-3ß and an increased protein content of Wnt3a, ß-catenin, annexin A1, and cyclin D1. After intervention with BXD, the body weight of the mice increased; the DAI score decreased; the colon length increased, and the tumor decreased. The histopathology showed reduced tumor proliferation and reduced inflammatory infiltration. The levels of IL-6 and IL-4 in the serum were significantly decreased, while the IL-2 content was increased. Meanwhile, the expression of E-cadherin was upregulated, and that of Ki67 and ß-catenin was downregulated. The protein content of E-cadherin and GSK-3ß increased, while that of Wnt3a, ß-catenin, annexin A1, and cyclin D1 decreased. In conclusion, BXD can inhibit CAC infected with Fn, and its potential mechanism may be related to the inhibition of Fn binding to E-cadherin, the decrease in annexin A1 protein level, and the regulation of the Wnt/ß-catenin pathway.

α-synuclein regulates Cyclin D1 to promote abnormal initiation of the cell cycle and induce apoptosis in dopamine neurons.

The etiology of Parkinson's disease (PD) is characterized by the death of dopamine neurons in the substantia nigra pars compacta, while misfolding and abnormal aggregation of α-synuclein (α-syn) are core pathological features. Previous studies have suggested that damage to dopamine neurons may be related to cell cycle dysregulation, but the specific mechanisms remain unclear. In this study, a PD mouse model was induced by stereotactic injection of α-syn into the nucleus, and treated with the cell cycle inhibitor, roscovitine (Rosc). The results demonstrated that Rosc improved behavioral disorders caused by α-syn, increased TH protein expression, inhibited α-syn and p-α-syn protein expression, and reduced the expression levels of G1/S phase cell cycle genes Cyclin D1, Cyclin E, CDK2, CDK4, E2F and pRB. Additionally, Rosc decreased Bax and Caspase-3 expression caused by α-syn, while increasing Bcl-2 protein expression. Meanwhile, we observed that α-syn can influence neuronal cell autophagy by decreasing the expression level of Beclin 1 and increasing the expression level of P62. However, Rosc can improve this phenomenon. In a cell model induced by α-syn in dopamine neuron injury cells, knockdown of Cyclin D1 led to similar results as those observed in animal experiments: Knocking down Cyclin D1 improved the abnormal initiation of the cell cycle caused by α-syn and regulated cellular autophagy, resulting in a reduction of apoptosis in dopamine neurons. In summary, exogenous α-syn can lead to the accumulation of α-syn and phosphorylated α-syn in dopamine neurons, increase key factors of the G1/S phase cell cycle such as Cyclin D1, and regulate downstream related indicators, causing the cell cycle to restart and leading to apoptosis of dopamine neurons. This exacerbates PD symptoms. However, knockdown of Cyclin D1 inhibits the progression of the cell cycle and can reverse this situation. These findings suggest that a Cyclin D inhibitor may be a novel therapeutic target for treating PD.

FAK mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation by modulating mitochondrial transcription termination factor 1/cyclin D1.

This study aimed to investigate the mechanism of FAK-dependent hypoxia-induced proliferation on human pulmonary artery smooth muscle cells (HPASMCs). Primary HPASMCs were isolated and cultured in vitro under normal and hypoxia conditions to assess cell proliferation with cell counting kit-8. FAK and mitochondrial transcription termination factor 1 (mTERF1) were silenced with siRNA, mRNA, and protein levels of FAK, mTERF1, and cyclin D1 were determined. HPASMC proliferation increased under hypoxia compared to normal conditions. Knocking down FAK or mTERF1 with siRNA led to decreased cell proliferation under both normal and hypoxia conditions. FAK knockdown led to the reduction of both mTERF1 and cyclin D1 expressions under the hypoxia conditions, whereas mTERF1 knockdown led to the downregulation of cyclin D1 expression but not FAK expression under the same condition. However, under normal conditions, knocking down either FAK or mTERF1 had no impact on cyclin D1 expression. These results suggested that FAK may regulate the mTERF1/cyclin D1 signaling pathway to modulate cell proliferation in hypoxia.

Gallic acid ameliorates endometrial hyperplasia through the inhibition of the PI3K/AKT pathway and the down-regulation of cyclin D1 expression.

BACKGROUND: Gallic acid (GA) is an organic compound with phenolic properties that occurs naturally and can be found in Guizhi Fuling capsules, showcasing a wide range of biological functionalities. PURPOSE: The objective of this study was to examine the influence of GA on endometrial hyperplasia (EH) and elucidate its underlying mechanism. METHODS: Initially, the induction of EH was achieved by administering estradiol to mice via continuous subcutaneous injection for a duration of 21 days. Concurrently, GA treatment was administered, and subsequently, the uterine tissue structure was assessed using hematoxylin and eosin (H&E) staining. Following this, the proliferation of human endometrial cells treated by GA was determined utilizing the CCK-8 method. Furthermore, network pharmacology and single-cell-RNA-seq data were employed to identify the target of GA action. In addition, we will employ immunofluorescence (IF), immunohistochemistry (IHC), flow cytometry, western blot and RT-qPCR methodologies to investigate the impact of GA on the expression level of cyclin D1, PI3K, p-PI3K, AKT, p-AKT. RESULTS: GA treatment ameliorated histopathological alterations in the uterus and suppress proliferation. Estradiol stimulation can activate the PI3K/AKT pathway, leading to up-regulation of cyclin D1 expression, whereas GA treatment results in down-regulation of its expression. CONCLUSIONS: The expression of cyclin D1 is down-regulated by GA through the inhibition of the PI3K/AKT pathway, effectively mitigating estradiol-induced EH in mice.

Combined SET7/9 and CDK4 inhibition act synergistically against osteosarcoma.

Osteosarcoma is the most common malignant bone tumor. It has a poor prognosis because of a lack of therapeutic targets and strategies. The SET domain-containing lysine-specific methyltransferase, SET7/9, has various functions in different cancer types in tissue-type and signaling context-dependent manners. The role of SET7/9 in osteosarcoma cells is currently controversial and its potential as a therapeutic candidate in osteosarcoma is unknown. In the present study, SET7/9 inhibition or ablation suppressed osteosarcoma cell proliferation by causing G1 arrest. Mechanistically, SET7/9 inhibition disrupted the interaction between cyclin-dependent kinase 4 (CDK4) and cyclin D1, which affected CDK4-cyclin D1 complex function, leading to decreased phosphorylation of retinoblastoma protein. CDK4 was overexpressed in osteosarcoma tissues and was closely related to a poor prognosis in patients with osteosarcoma. We therefore hypothesized that SET7/9 inhibition might increase the sensitivity of osteosarcoma cells to CDK4 inhibitors, potentially decreasing the risk of adverse effects of CDK4 inhibitors. The combination of SET7/9 and CDK4 inhibition enabled dose reductions of both inhibitors and had a synergistic effect against osteosarcoma growth in vivo. Collectively, these findings indicate that SET7/9 plays an oncogenic role in osteosarcoma by regulating CDK4-cyclin D1 complex interaction and function. The combination of SET7/9 and CDK4 inhibition may thus provide a novel effective therapeutic strategy for osteosarcoma with no significant toxicity.

LncRNA FOXD3-AS1 Contributes to Glioblastoma Progression Via Sponging miR-3918 to Upregulate CCND1.

AIM: To elucidate the pro-tumorigenic role of IncRNA FOXD3-AS1 in glioblastoma. MATERIAL AND METHODS: The expression of miR-3918, FOXD3-AS1, and CCND1 was measured in glioblastoma cells and tissues using reverse transcriptase quantitative PCR (RT-qPCR). The effect of FOXD3-AS1 silencing on the proliferation of glioblastoma cells was assessed in vitro using CCK-8 and colony formation assays and in vivo using xenograft mouse models. Additionally, the expression levels of the apoptosis-related proteins, Bcl-2 and Bax, were assessed using western blotting. Bioinformatic analysis and luciferase reporter assays assisted by RNA immunoprecipitation (RIP) and RNA pull-down experiments were conducted to validate the interactions among FOXD3-AS1, CCND1, and miR-3918. RESULTS: FOXD3-AS1 and CCND1 were highly expressed in glioblastoma tissues and cells, whereas miR-3918 was poorly expressed. The expressions of FOXD3-AS1 and CCND1 were inversely associated with miR-3918 levels in glioblastoma tissues. FOXD3-AS1 silencing weakened the proliferative capacity and accelerated apoptosis of glioblastoma cells in vitro and hampered tumor growth in vivo. Mechanical investigations showed that FOXD3-AS1 knockdown increased miR-3918 expression and inhibited glioblastoma cell growth. Meanwhile, the miR-3918 inhibitor restored CCND1 expression and induced the opposite outcome. CONCLUSION: FOXD3-AS1 facilitates the CCND1-driven progression of glioblastoma by serving as a competing endogenous RNA (ceRNA) for miR-3918. This suggests that FOXD3-AS1 may be a potential therapeutic target for the management of glioblastoma development.

Cisplatin-resistance induces lung squamous carcinoma cell growth by nicotine-mediated α7nAchR/HDAC1/Cyclin D1/pRb cell cycle activation.

The majority of adenocarcinoma lung cancer is found in nonsmokers. A history of tobacco use is more common in squamous cell carcinoma of the lung. The aim of this study is to identify the cisplatin (CDDP)-resistance that promotes lung squamous carcinoma cell growth through nicotine-mediated HDAC1/7nAchR/E2F/pRb cell cycle activation. Squamous cell carcinoma (NCI-H520 and NCI-H157) cells were examined after cisplatin and nicotine treatment by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay, cell migration assay, immunofluorescence staining, western blot analysis, and immunoprecipitation analysis. Consequently, CDDP is released from DNA and Rb phosphorylated pRb as a result of nicotine-induced cancer cell proliferation through 7nAchR, which then triggers the opening of the HDAC1 cell cycle. The cell cycle is stopped when CDDP adducts are present. Nicotine exerts cancer cytoprotective effects by allowing HDAC1 repair mechanisms to re-establish E2F promoting DNA stimulation cell cycle integrity in the cytosol and preventing potential CDDP and HDAC1 suppressed in the nuclear. Concentration expression of nicotine causes squamous carcinoma cell carcinogens to emerge from inflammation. COX2, NF-KB, and NOS2 increase as a result of nicotine-induced squamous carcinoma cell inflammation. Nicotine enhanced the cell growth-related proteins such as α7nAchR, EGFR, HDAC1, Cyclin D, Cyclin E, E2F, Rb, and pRb by western blot analysis. It also induced cancer cell inflammation and growth. As a result, we suggest that nicotine will increase the therapeutic resistance effects of CDDP. This has the potential to interact with nicotine through α7nAchR receptors and HDAC1/Cyclin D/E2F/pRb potentially resulting in CDDP therapy resistance, as well as cell cycle-induced cancer cell growth.

NEDD4L is a promoter for angiogenesis and cell proliferation in human umbilical vein endothelial cells.

Dysregulated angiogenesis leads to neovascularization, which can promote or exacerbate various diseases. Previous studies have proved that NEDD4L plays an important role in hypertension and atherosclerosis. Hence, we hypothesized that NEDD4L may be a critical regulator of endothelial cell (EC) function. This study aimed to define the role of NEDD4L in regulating EC angiogenesis and elucidate their underlying mechanisms. Loss- and gain-of-function of NEDD4L detected the angiogenesis and mobility role in human umbilical vein endothelial cells (HUVECs) using Matrigel tube formation assay, cell proliferation and migration. Pharmacological pathway inhibitors and western blot were used to determine the underlying mechanism of NEDD4L-regulated endothelial functions. Knockdown of NEDD4L suppressed tube formation, cell proliferation and cell migration in HUVECs, whereas NEDD4L overexpression promoted these functions. Moreover, NEDD4L-regulated angiogenesis and cell progression are associated with the phosphorylation of Akt, Erk1/2 and eNOS and the expression of VEGFR2 and cyclin D1 and D3. Mechanically, further evidence was confirmed by using Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Overexpression NEDD4L-promoted angiogenesis, cell migration and cell proliferation were restrained by these inhibitors. In addition, overexpression NEDD4L-promoted cell cycle-related proteins cyclin D1 and D3 were also suppressed by Akt blocker MK-2206, Erk1/2 blocker U0126 and eNOS blocker L-NAME. Our results demonstrated a novel finding that NEDD4L promotes angiogenesis and cell progression by regulating the Akt/Erk/eNOS pathways.

G-quadruplexes promote the motility in MAZ phase-separated condensates to activate CCND1 expression and contribute to hepatocarcinogenesis.

G-quadruplexes (G4s) can recruit transcription factors to activate gene expression, but detailed mechanisms remain enigmatic. Here, we demonstrate that G4s in the CCND1 promoter propel the motility in MAZ phase-separated condensates and subsequently activate CCND1 transcription. Zinc finger (ZF) 2 of MAZ is a responsible for G4 binding, while ZF3-5, but not a highly disordered region, is critical for MAZ condensation. MAZ nuclear puncta overlaps with signals of G4s and various coactivators including BRD4, MED1, CDK9 and active RNA polymerase II, as well as gene activation histone markers. MAZ mutants lacking either G4 binding or phase separation ability did not form nuclear puncta, and showed deficiencies in promoting hepatocellular carcinoma cell proliferation and xenograft tumor formation. Overall, we unveiled that G4s recruit MAZ to the CCND1 promoter and facilitate the motility in MAZ condensates that compartmentalize coactivators to activate CCND1 expression and subsequently exacerbate hepatocarcinogenesis.

Overexpression of Alpha-1 Antitrypsin Increases the Proliferation of Mesenchymal Stem Cells by Upregulation of Cyclin D1.

Alpha-1 antitrypsin-overexpressing mesenchymal stromal/stem cells (AAT-MSCs) showed improved innate properties with a faster proliferation rate when studied for their protective effects in mouse models of diseases. Here, we investigated the potential mechanism(s) by which AAT gene insertion increases MSC proliferation. Human bone marrow-derived primary or immortalized MSCs (iMSCs) or AAT-MSCs (iAAT-MSCs) were used in the study. Cell proliferation was measured by cell counting and cell cycle analysis. Possible pathways involved in the pro-proliferation effect of AAT were investigated by measuring mRNA and protein expression of key cell cycle genes. Interval cell counting showed increased proliferation in AAT-MSCs or iAAT-MSCs compared to their corresponding MSC controls. Cell cycle analysis revealed more cells progressing into the S and G2/M phases in iAAT-MSCs, with a notable increase in the cell cycle protein, Cyclin D1. Moreover, treatment with Cyclin D1 inhibitors showed that the increase in proliferation is due to Cyclin D1 and that the AAT protein is upstream and a positive regulator of Cyclin D1. Furthermore, AAT's effect on Cyclin D1 is independent of the Wnt signaling pathway as there were no differences in the expression of regulatory proteins, including GSK3ß and ß-Catenin in iMSC and iAAT-MSCs. In summary, our results indicate that AAT gene insertion in an immortalized MSC cell line increases cell proliferation and growth by increasing Cyclin D1 expression and consequently causing cells to progress through the cell cycle at a significantly faster rate.

Vitamin D improves autoimmune diseases by inhibiting Wnt signaling pathway.

OBJECTIVE: In this study, we investigated the development of the Wnt signaling pathway in vitamin D (VitD) to improve systemic lupus erythematosus in mice to breakthrough clinical treatment approaches. METHODS: Body weight changes were recorded during rearing. Antinuclear antibodies (ANA), anti-dsDNA, and anti-snRNP were detected in the mouse serum using an enzyme-linked immunosorbent assay. Apoptosis of Th1 and Th2 immune cells in mice was detected using flow cytometry. Reverse transcription polymerase chain reaction was used to detect the expression of T-bet, GATA3, and Wnt3a mRNA in the spleens of each group. Western blot analysis was performed to detect the expression of Wnt1, p-ß-catenin, ß-catenin, glycogen synthase kinsase3ß (GSK-3ß), Wnt3a, c-myc, and cyclin D1 protein in mice spleens. ß-catenin in mice spleen was visualized using immunohistochemistry. RESULTS: VitD did not substantial reduce the body weight of MRL/LPR mice, whereas the inhibitor did. VitD notably decreased the concentrations of ANA, anti-double-stranded DNA, and anti-snRNP in the serum of MRL/LPR mice and alleviated apoptosis of Th1 and Th2 cells. VitD markedly increased the expression of T-bet and GATA mRNA in the spleen of MRL/LPR mice and consequently increased the levels of Wnt3a and ß-catenin. Western blot analysis revealed that the levels of GSK-3ß, p-ß-catenin, Wnt1, Wnt3a, c-myc, and cyclin D1 could be reduced by VitD, compared with MRL/LPR. Immunohistochemistry demonstrated that the expression of ß-catenin was the most pronounced in the spleen of MRL/LPR mice, and the expression level of ß-catenin decreased substantially after VitD intervention. CONCLUSIONS: VitD can further inhibit the nuclear translocation of ß-catenin by downregulating the expression of Wnt ligands (Wnt1 and Wnt3a), which reduces the expression of the downstream target gene cyclin D1. Systemic lupus erythematosus in mice was improved by inhibiting the activation of Wnt/ß-catenin signal pathway.

PC4: A new regulator of cyclin D1 transcript levels.

The expression of cyclin proteins is tightly regulated during the cell cycle, to allow precise activation of cyclin-dependent kinases. In this issue, Pan et al. (https://doi.org/10.1083/jcb.202308066) identify an RNA-binding protein, PC4, as a regulator of cyclin D1 mRNA stability in hepatocellular carcinoma cells. This study provides a new mechanism regulating the levels of a key cell cycle protein, cyclin D1, in human cells.

Inhibition of amino acid transporter LAT1 in cancer cells suppresses G0/G1-S transition by downregulating cyclin D1 via p38 MAPK activation.

L-type amino acid transporter 1 (LAT1, SLC7A5) is upregulated in various cancers and associated with disease progression. Nanvuranlat (Nanv; JPH203, KYT-0353), a selective LAT1 inhibitor, suppresses the uptake of large neutral amino acids required for rapid growth and proliferation of cancer cells. Previous studies have suggested that the inhibition of LAT1 by Nanv induces the cell cycle arrest at G0/G1 phase, although the underlying mechanisms remain unclear. Using pancreatic cancer cells arrested at the restriction check point (R) by serum deprivation, we found that the Nanv drastically suppresses the G0/G1-S transition after release. This blockade of the cell cycle progression was accompanied by a sustained activation of p38 mitogen-activated protein kinase (MAPK) and subsequent phosphorylation-dependent proteasomal degradation of cyclin D1. Isoform-specific knockdown of p38 MAPK revealed the predominant contribution of p38α. Proteasome inhibitors restored the cyclin D1 amount and released the cell cycle arrest caused by Nanv. The increased phosphorylation of p38 MAPK and the decrease of cyclin D1 were recapitulated in xenograft tumor models treated with Nanv. This study contributes to delineating the pharmacological activities of LAT1 inhibitors as anti-cancer agents and provides significant insights into the molecular basis of the amino acid-dependent cell cycle checkpoint at G0/G1 phase.

Molecular mechanism of triptolide in myocardial fibrosis through the Wnt/ß-catenin signaling pathway.

Objective. Myocardial fibrosis (MF) is a common manifestation of end-stage cardiovascular diseases. Triptolide (TP) provides protection against cardiovascular diseases. This study was to explore the functional mechanism of TP in MF rats via the Wnt/ß-catenin pathway. Methods. The MF rat model was established via subcutaneous injection of isoproterenol (ISO) and treated with low/medium/high doses of TP (L-TP/M-TP/H-TP) or Wnt agonist BML-284. Cardiac function was examined by echocardiography. Pathological changes of myocardial tissues were observed by HE and Masson staining. Col-I/Col-III/Vimentin/α-SMA levels were detected by immunohistochemistry, RT-qPCR, and Western blot. Collagen volume fraction content was measured. Expression levels of the Wnt/ß-catenin pathway-related proteins (ß-catenin/c-myc/Cyclin D1) were detected by Western blot. Rat cardiac fibroblasts were utilized for in vitro validation experiments. Results. MF rats had enlarged left ventricle, decreased systolic and diastolic function and cardiac dysfunction, elevated collagen fiber distribution, collagen volume fraction and hydroxyproline content. Levels of Col-I/Col-III/Vimentin/α-SMA, and protein levels of ß-catenin/c-myc/Cyclin D1 were increased in MF rats. The Wnt/ß-catenin pathway was activated in the myocardial tissues of MF rats. TP treatment alleviated impairments of cardiac function and myocardial tissuepathological injury, decreased collagen fibers, collagen volume fraction, Col-I, Col-III, α-SMA and Vimentin levels, HYP content, inhibited Wnt/ß-catenin pathway, with H-TP showing the most significant effects. Wnt agonist BML-284 antagonized the inhibitive effect of TP on MF. TP inhibited the Wnt/ß-catenin pathway to repress the proliferation and differentiation of mouse cardiac fibroblasts in vitro. Conclusions. TP was found to ameliorate ISO-induced MF in rats by inhibiting the Wnt/ß-catenin pathway.

Unveiling the therapeutic potential of thymol from Nigella sativa L. seed: selective anticancer action against human breast cancer cells (MCF-7) through down-regulation of Cyclin D1 and proliferative cell nuclear antigen (PCNA) expressions.

BACKGROUND: Breast adenocarcinoma cells (MCF-7) are characterized by the overexpression of apoptotic marker genes and proliferative cell nuclear antigen (PCNA), which promote cancer cell proliferation. Thymol, derived from Nigella sativa (NS), has been investigated for its potential anti-proliferative and anticancer properties, especially its ability to suppress Cyclin D1 and PCNA expression, which are crucial in the proliferation of cancer cells. METHODS: The cytotoxicity of thymol on MCF-7 cells was assessed using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) release methods. Thymol was tested at increasing concentrations (0-1000 µM) to evaluate its impact on MCF-7 cell growth. Additionally, Cyclin D1 and PCNA gene expression in thymol-treated and vehicle control groups of MCF-7 were quantified using real-time Polymerase Chain Reaction (RT-qPCR). Protein-ligand interactions were also investigated using the CB-Dock2 server. RESULTS: Thymol significantly inhibited MCF-7 cell growth, with a 50% inhibition observed at 200 µM. The gene expression of Cyclin D1 and PCNA was down-regulated in the thymol-treated group relative to the vehicle control. The experimental results were verified through protein-ligand interaction investigations. CONCLUSIONS: Thymol, extracted from NS, demonstrated specific cytotoxic effects on MCF-7 cells by suppressing the expression of Cyclin D1 and PCNA, suggesting its potential as an effective drug for MCF-7. However, additional in vivo research is required to ascertain its efficacy and safety in medical applications.

The effect and mechanism of low-molecular-weight heparin on the decidualization of stromal cells in early pregnancy.

OBJECTIVE: This study aimed to analyze the effect of low-molecular-weight heparin (LMWH) on the decidualization of stromal cells in early pregnancy and explore the effect of LMWH on pregnancy outcomes. METHODS: Recurrent spontaneous abortion (RSA) mouse model (CBA/J × DBA/2) and normal pregnant mouse model (CBA/J × BALB/c) were established. The female mice were checked for a mucus plug twice daily to identify a potential pregnancy. When a mucus plug was found, conception was considered to have occurred 12 h previously. The pregnant mice were divided randomly into a normal pregnancy control group, an RSA model group, and an RSA + LMWH experimental group (n = 10 mice in each group). Halfway through the 12th day of pregnancy, the embryonic loss of the mice was observed; a real-time quantitative polymerase chain reaction was used to detect the messenger ribonucleic acid (mRNA) expressions of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) in the decidua of the mice. Additionally, the decidual tissues of patients with RSA and those of normal women in early pregnancy who required artificial abortion were collected and divided into an RSA group and a control group. Decidual stromal cells were isolated and cultured to compare cell proliferation between the two groups, and cellular migration and invasion were detected by membrane stromal cells. Western blotting was used to detect the protein expressions of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase- (MMP) 2, and MMP-7 in stromal cells treated with LMWH. RESULTS: Compared with the RSA group, LMWH significantly reduced the pregnancy loss rate in the RSA mice (p < 0.05). Compared with the RSA group, the LMWH + RSA group had significantly higher expression levels of PRL and IGFBP1 mRNA (p < 0.01). LMWH promoted the proliferation, migration, and invasion of human decidual stromal cells; compared with the control group, the expression levels of MMP-2, MMP-7, cyclin D1, and PCNA proteins in the decidual stromal cells of the LMWH group increased (p < 0.05). CONCLUSIONS: The use of LMWH can improve pregnancy outcomes by enhancing the proliferation and migration of stromal cells in early pregnancy and the decidualization of stromal cells.

Discovery of a small molecule that inhibits Bcl-3-mediated cyclin D1 expression in melanoma cells.

Molecular targeted therapy using a drug that suppresses the growth and spread of cancer cells via inhibition of a specific protein is a foundation of precision medicine and treatment. High expression of the proto-oncogene Bcl-3 promotes the proliferation and metastasis of cancer cells originating from tissues such as the colon, prostate, breast, and skin. The development of novel drugs targeting Bcl-3 alone or in combination with other therapies can cure these patients or prolong their survival. As a proof of concept, in the present study, we focused on metastatic melanoma as a model system. High-throughput screening and in vitro experiments identified BCL3ANT as a lead molecule that could interfere with Bcl-3-mediated cyclin D1 expression and cell proliferation and migration in melanoma. In experimental animal models of melanoma, it was demonstrated that the use of a Bcl-3 inhibitor can influence the survival of melanoma cells. Since there are no other inhibitors against Bcl-3 in the clinical pipeline for cancer treatment, this presents a unique opportunity to develop a highly specific drug against malignant melanoma to meet an urgent clinical need.

Mechanistic study on ursolic acid inhibiting the growth of colorectal cancer cells through the downregulation of TGF-ß3 by miR-140-5p.

Colorectal cancer (CRC) is a common digestive tract tumor with a high incidence and a poor prognosis. Traditional chemotherapy drugs are usually accompanied by unpleasant side effects, highlighting the importance of exploring new adjunctive drugs. In this study, we aimed to explore the role of ursolic acid (UA) in CRC cells. Specifically, HT-29 cells were treated with UA at different concentrations (10, 20, 30, and 40 µM), and the expression of miR-140-5p, tumor growth factor-ß3 (TGF-ß3), ß-catenin, and cyclin D1 was determined by real-time quantitative PCR. The cell cycle and apoptosis were checked by flow cytometry, and cell proliferation was detected by Cell Counting Kit-8 assay. The HT-29 cell model was established through overexpression (miR-140-5p mimics) and interference (miR-140-5p inhibitor) of miR-140-5p. Western blot was used to detect the protein expression of TGF-ß3. We found that UA could inhibit the proliferation of HT-29 cells, block cells in the G1 phase, and promote cell apoptosis. After UA treatment, the expression of miR-140-5p increased and TGF-ß3 decreased. Notably, miR-140-5p downregulated the expression of TGF-ß3, while the overexpression of miR-140-5p exerted a similar function to UA in HT-29 cells. Additionally, the messenger RNA expression of TGF-ß3, ß-catenin, and cyclin D1 was decreased in HT-29 cells after UA treatment. In conclusion, UA inhibited CRC cell proliferation and cell cycle and promoted apoptosis by regulating the miR-140-5p/TGF-ß3 axis, which may be related to the inhibition of Wnt/ß-catenin signaling pathway.

Enhanced anticancer synergy of LOM612 in combination with selinexor: FOXO1 nuclear translocation-mediated inhibition of Wnt/ß-catenin signaling pathway in breast cancer.

BACKGROUND: The intricate relationship between Forkhead box O1 (FOXO1), a well-established tumor suppressor, and breast cancer (BC) remains partially elucidated. This study aims to investigate the mechanistic role of FOXO1 nuclear localization in the context of BC. METHODS: In vitro experiments employed BC cell lines MCF-7 and MDA-MB-175 treated with LOM612, a small molecule activator of FOXO nuclear-cytoplasmic shuttling, and selinexor, an exportin 1 inhibitor. Nuclear accumulation of FOXO1, its interaction with ß-catenin, and expressions of key proteins like V-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), cyclin D1 and apoptosis markers were assessed. In vivo, the effects of LOM612 and selinexor were studied using MCF-7 cell-derived xenografts (CDX). RESULTS: Treatment with LOM612 exhibited a significant enhancement in nuclear accumulation of FOXO1 within BC cells. This effect coincided with suppressed migratory behavior and heightened apoptosis susceptibility in these cells. Mechanistically, LOM612 orchestrated FOXO1 to compete with transcription factors (TCF) for binding to ß-catenin in the nucleus, leading to reduced c-Myc and cyclin D1 expressions, along with elevated levels of apoptosis-related proteins. Similar trends were observed in CDX models, where LOM612 effectively suppressed tumor growth, increased FOXO1 nuclear localization, and downregulated c-Myc and cyclin D1 expressions. Importantly, selinexor synergistically reinforced the therapeutic effects of LOM612 both in vitro and in vivo. CONCLUSIONS: Collectively, this study underscores the potential of combining LOM612 and selinexor as an efficacious anti-BC strategy. The underlying mechanism involves FOXO1's nuclear translocation, which disrupts TCF-ß-catenin interactions, thus indirectly inhibiting the Wnt/ß-catenin signaling pathway.