Cyclin D1 Research Articles

Focal adhesion kinase promotes aerobic glycolysis in hepatic stellate cells via the cyclin D1/c-Myc/MCT-1 pathway to induce liver fibrosis

Hepatic stellate cells (HSCs) transdifferentiate into myofibroblasts during liver fibrosis and exhibit increased glycolysis. Phosphorylated focal adhesion kinase (FAK) (pY397-FAK) promotes monocarboxylate transporter 1 (MCT-1) expression in HSCs to increase aerobic glycolysis and cause liver fibrosis. A combined multiomics analysis of C57BL/6 mice with tetrachloromethane (CCl4)-induced liver fibrosis was performed to identify the downstream FAK signaling pathway. The effect of the FAK inhibitor PF562271 on CCl4-induced liver fibrosis was explored by immunofluorescence of liver tissues. The migration, proliferation and aerobic glycolysis of LX-2 cells after stimulation and activation by transforming growth factor beta-1 (TGF-β1) or suppression by PF562271 was assessed in vitro. Multiomics analysis of a successfully generated CCl4-induced liver fibrosis mouse model was performed. FAK and cyclin D1 were significantly enriched in mice with CCl4-induced liver fibrosis. In vivo, the MCT-1 and alpha smooth muscle actin (α-SMA) levels were increased in mice with CCl4-induced liver fibrosis, and MCT-1 and α-SMA expression decreased after PF562271 treatment. In vitro, PF562271 alleviated TGF-β1-induced LX-2 activation. LX-2 cells showed diminished migration, proliferation, and aerobic glycolysis after PF562271 intervention. FAK promotes aerobic glycolysis in LX-2 cells through the cyclin D1/c-Myc/MCT-1 pathway, thereby increasing liver fibrosis.

RNF6 inhibits lung adenocarcinoma cell proliferation by promoting cyclin D2 degradation.

The E3 ubiquitin ligase RNF6 has been widely recognized for its role in promoting tumorigenesis in multiple cancers. However, we found it is downregulated in lung adenocarcinoma (LUAD) and the molecular rationale for this discrepancy remains unclear. In the present study, we find that RNF6 but not its ΔRING inactive form inhibits LUAD cell proliferation and migration and sensitizes LUAD to chemotherapy. To understand the molecular mechanism, we utilize affinity purification/tandem mass spectrometry to analyze RNF6-interacting proteins and find that cyclin D2 (CCND2), a key regulator of the G1/S transition in the cell cycle. RNF6 physically binds to CCND2 and mediates its K48-linked polyubiquitination and subsequent degradation. However, ΔRING RNF6 fails to mediate CCND2 for ubiquitination and degradation. Moreover, Thr280 is critically important for CCND2 stability. When Thr280 is mutated, CCND2 becomes more stable and less ubiquitinated by RNF6. Furthermore, RNF6 arrests LUAD cell cycle at the G1 phase by inhibiting the CCND2/pRb signaling pathway, which is consistent with decreased cell proliferation. Lastly, RNF6 curtails the growth of LUAD xenografts in vivo, associated with decreased CCND2 expression. Therefore, RNF6 is a novel E3 ligase of CCND2 and suppresses LUAD cell proliferation. Implications: This study reveals a novel regulation on cell cycle transition in LUAD and suggests the RNF6/CCND2 axis may represent an alternative therapeutic target for the treatment of LUAD.

The Role of Senescence in Experimental Periodontitis at the Causal Level: An in Vivo Study

The occurrence and severity of periodontitis (PD) tend to increase with age, and yet the underlying mechanisms remain unclear. Immune senescence is known to be triggered in mice and humans as they age. Experimental PD in mice has been shown to induce senescence biomarkers p16 INK4a and p21, dysfunction of antigen-presenting cells (APCs), and activation of the senescence-associated secretory phenotype (SASP). However, the causal links of senescence to experimental PD are not yet established. This study aims to elucidate the role of senescence in experimental PD at a causal level. The P16-3MR mouse model harbors the p16INK4a (Cdkn2a) promoter, driving in vivo expression of synthetic Renilla luciferase, monomeric red fluorescent protein (mRFP), and herpes simplex virus-1 thymidine kinase (HSV-TK). This facilitates in vivo identification of p16 INK4a activation at the cellular level and the consequences of selective elimination of p16INK4a-positive cells by ganciclovir (GCV) treatment. Mice were treated with/without GCV for two weeks during ligature-induced PD. In vivo bioluminescence imaging quantified p16INK4a activation, while Western blot and immunofluorescence analyses assessed key senescence and inflammatory markers (p16, p21, p53, Cyclin D1, p-H2A.X, IL17, and IL1β). Alveolar bone volume was analyzed by micro-CT and histomorphometry. Our findings demonstrate that clearance of senescent cells in mice subjected to experimental PD alleviates inflammation and mitigates bone loss. These results suggest a causal role for senescence in PD pathology, raising the future prospect of senolytic agents for therapeutic intervention in PD.

Fibrinopeptide a promotes the proliferation and migration of vascular smooth muscle cells by regulating the integrin αVβ3/PI3K/AKT signaling pathway.

Atherosclerosis is characterized by subintimal proliferation and migration of vascular smooth muscle cells (VSMCs) in response to stimuli such as coagulation and inflammatory factors. Fibrinopeptide A (FPA), a biomarker for coagulation system activation, is elevated in patients with ischemic heart disease. However, its role in the pathophysiology of cardiovascular disorders remains unclear. This study aimed to investigate the impact of FPA on VSMCs proliferation and migration and elucidate the underlying molecular mechanisms. Transcriptome sequencing and bioinformatics analysis were employed to elucidate molecular pathways. Scratch wound and Transwell assays were performed to evaluate cell migration capacity. Molecular expression patterns were assessed using immunofluorescence, real-time quantitative PCR, and Western blot assays. The differentially expressed genes (DEGs) in the FPA-treated VSMCs were enriched in the cell cycle and PI3K/AKT signaling pathway. FPA treatment enhanced VSMCs' migratory capacity and upregulated integrin αVβ3, PI3K, P-AKT, AKT, Cyclin D1, and PCNA expression. The integrin αVβ3 inhibitor Cyclo-RGDfk effectively suppressed VSMCs migration and reduced the expression levels of these genes in FPA-stimulated VSMCs. These results suggested that FPA promotes the proliferation and migration of VSMCs by regulating the PI3K/AKT signaling pathway through integrin αVβ3.

Carthamin yellow-loaded glycyrrhetinic acid liposomes alleviate interstitial fibrosis in diabetic nephropathy

Objectives To investigate the therapeutic efficacy of Carthamin yellow (CY)-loaded glycyrrhetinic acid (GA) liposomes in treating diabetic nephropathy (DN), particularly in alleviating renal interstitial fibrosis and improving kidney function. Methods CY-loaded GA liposomes were prepared and characterized for structural stability and controlled release. DN rat models were treated with CY-loaded GA liposomes, and kidney pathology, function, collagen deposition, and TGF-β1 expression were evaluated. The effects of CY-loaded GA liposomes were compared to Vitamin E and CY alone. In vitro experiments with TGF-β1-stimulated human renal interstitial fibroblasts (hRIFs) examined the effects of CY-loaded GA liposomes on cell proliferation and the expression of fibrotic markers. Mechanistic studies assessed the role of the TGFBR1/Smad2/Smad3 pathway using TGFBR1 overexpression experiments. Results The CY-loaded GA liposomes exhibited a stable structure and controlled release profile. In DN rats, treatment with CY-loaded GA liposomes significantly alleviated kidney damage, improved kidney function, reduced collagen deposition and fibrosis, and downregulated TGF-β1 expression, showing superior effects compared to Vitamin E or CY alone. In TGF-β1-stimulated hRIFs, CY-loaded GA liposomes effectively suppressed cell proliferation and reduced the expression of Cyclin D1, PCNA, fibronectin, and collagen I. The inhibitory effects were stronger than CY alone and were mediated by the inactivation of the TGFBR1/Smad2/Smad3 pathway, as confirmed by TGFBR1 overexpression studies. Conclusions CY-loaded GA liposomes demonstrated significant therapeutic efficacy in alleviating renal interstitial fibrosis in DN by targeting the TGFBR1/Smad2/Smad3 pathway. This novel drug delivery system provides a promising approach for the treatment of DN.

Identifying CSNK1E as a therapeutic target in thyroid cancer among the core circadian clock genes.

Previous studies have shown that thyroid malignancies can alter the transcriptional oscillations of circadian clock genes. In this study, we screened the expression of core circadian clock genes in thyroid neoplasms and found that CSNK1E, NPAS2, and TIMELESS were upregulated, while ARNTL, CRY1, CRY2, PER2, and RORA were downregulated during the progression and dedifferentiation of thyroid cancer. Immunohistochemical analysis further confirmed an increase in CSNK1E expression parallel to the loss of tumor differentiation. To investigate the potential therapeutic implications, we treated thyroid cancer cell lines with two different CSNK1E inhibitors: PF670462 and IC261. Both inhibitors resulted in growth inhibition in monolayer and three-dimensional spheroid cultures. This growth inhibition was accompanied by G2/M cell cycle arrest and a decrease in CDK4 and cyclin D1 expression. Moreover, CSNK1E inhibitors suppressed cell migration and invasion and reduced the expression of epithelial-mesenchymal transition markers. In vivo experiments using xenograft models showed that the administration of IC261 significantly restrained tumor growth and decreased the Ki-67 index of the xenograft tumors. In conclusion, our study provides evidence of aberrant CSNK1E expression in thyroid cancer dedifferentiation and highlights the potential therapeutic value of targeting CSNK1E.

BRD4 Induces Esophageal Squamous Cell Carcinoma Progression via the Wnt/β-catenin Pathway.

BRD4, part of the bromodomain and extra terminal domain (BET) protein family, plays a pivotal role in gene transcription, DNA replication, and repair via transcription regulators. Despite its established involvement in various human diseases, its function in esophageal squamous cell carcinoma (ESCC) has not been fully explored. Our research investigated the association of BRD4 in ESCC and its underlying molecular mechanisms. The findings revealed that BRD4 knockdown notably diminished the cells' proliferation, migration, invasion capabilities and induced apoptosis and cell cycle arrest. Conversely, overexpression of BRD4 can reverse these phenotypes. Pearson correlation and enrichment analyses indicated that BRD4 expression was associated with the cell cycle and Wnt/β-catenin signaling pathway. Further validation confirmed that reduced BRD4 expression downregulates Cyclin D1 and c-Myc, and suppresses epithelial-to-mesenchymal transition (EMT) and Wnt/β-catenin signaling pathway. Furthermore, rescue experiments showed that overexpressing c-Myc significantly mitigated the inhibitory impact of BRD4. Moreover, by employing single-cell transcriptome sequencing, we explored the impact of the tumor microenvironment on BRD4 overexpression in ESCC cells. These insights confirmed BRD4's potential as a therapeutic target, suggesting that modulating its expression could yield promising strategies for ESCC treatment.

Dahuang-Gancao decoction ameliorates testosterone-induced androgenetic alopecia in mice.

Dahuang-Gancao decoction (DGD) is a traditional Chinese medicinal formula that is recorded in the Synopsis of the Golden Chamber, and is widely used to treat damp-heat in the body. Since the pathological factors of androgenetic alopecia (AGA) also reflect damp-heat blockage, DGD has great potential for the treatment of AGA and has been used effectively in clinical practice. The aim of the study was to investigate whether external application of DGD could promote the activation and proliferation of hair follicle stem cells (HFSCs) and improve AGA through the Wnt/β-catenin pathway. The main chemical components of DGD-contained serum were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database search. Cell Counting Kit-8 (CCK8) was used to investigate the appropriate concentration. Hair regeneration was assessed by hair growth score and histopathological staining. The proliferation of HFSCs and the activation of Wnt/β-catenin pathway were detected by Western blot, immunofluorescence staining, real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and enzyme-linked immunosorbent assay (ELISA). The AGA mouse model was induced by external application of testosterone (T). Immunofluorescence staining was performed to localize HFSCs by CK15, followed by staining with Ki67, β-catenin, and Cyclin D1, respectively. The results illustrated that the 10% DGD group and the 10% DGD+HLY78 group could significantly promote the expression of Wnt10b and β-catenin and the proliferation of HFSCs in vitro, while the 10% DGD+IWR-1 group could reverse the promotion effect of DGD. Animal experiments showed that compared with the model group (T group), DGD promoted hair follicles to enter the anagen phase, as evidenced by an increase in hair growth score, an increase in the number of hair follicles in hematoxylin and eosin (HE) staining, and a significant increase in the ratio of the number of anagen follicles to the total number of hair follicles (AF/AF+TF). In addition, DGD upregulated the expression of Wnt/β-catenin signaling pathway proteins in the skin tissues of AGA mice. It also promoted the proliferation of HFSCs and the expression of β-catenin and Cyclin D1 cytokines in the region of HFSCs. Both oral and external application of DGD can promote the proliferation of HFSCs by activating the Wnt/β-catenin signalling pathway. External application of DGD can promote the hair follicles to enter the anagen phase, which can ameliorate the symptoms of alopecia in AGA mice. Therefore, compared to oral DGD, external application of DGD is an effective and safer way of administration for the treatment of AGA.

Nimbolide attenuates hepatocellular carcinoma by regulating miRNAs 21, 145 and 221 and their target gene expression.

MicroRNAs (miRNAs) are becoming progressively emerging in cancer research from an etiologic and curative point of view. Several miRNAs act as oncogenes or tumor suppressors, which are dysregulated in numerous cancers. Our previous studies have established that nimbolide (a bioactive terpenoid from neem) attenuated hepatocellular carcinoma (HCC) through various mechanisms in mice. Here, we aimed to elucidate the effect of nimbolide in modulating specific miRNAs (21, 145, and 221) and their target genes involved in promoting inflammation and cancer cell proliferation in HCC mice. Following the induction of HCC in mice at 28weeks, nimbolide (6mg/kg b.wt.) was administered orally for four consecutive weeks. We found significantly increased hepatic expression of miR-21a-3p, miR-21a-5p, miR-221-5p and miR-221-3p whilst significantly decreased miR-145a-5p in HCC mice. Nimbolide treatment to HCC mice substantially reduced the miR-21a-5p and miR-221-3p and improved miR-145a-5p gene expression. Our in-silico study also supports these findings. Moreover, hepatic tight junction (TJ) associated proteins such as claudins 1&5 mRNA and protein were increased considerably, whilst significantly decreased hepatic claudin 2 mRNA and protein expression noted in HCC mice. Nimbolide also regulates cadherins, ROCK 1, MMP 9, cyclin D1, CDK4, NF κB and TNFα mRNA expression in HCC mice. We identified for the first time that nibmolide treatment to HCC mice significantly attenuated hepatic miRNAs 21 & 221 expressions and sheltered miR-145 expression. These findings were further confirmed with in-silico studies. Moreover, nibmolide treatment in HCC mice regulates miRNA target genes involved in cancer cell proliferation and inflammation, thereby attenuating HCC progression in mice.

Role of TNFRSF12A in cell proliferation, apoptosis, and proinflammatory cytokine expression by regulating the MAPK and NF-κB pathways in thyroid cancer cells.

Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A) has been reported to be upregulated in thyroid cancer (THCA). However, the role and mechanism of TNFRSF12A in THCA remain largely unknown. TNFRSF12A expression in THCA samples was analyzed using bioinformatics analysis. CCK-8, EdU incorporation assay, TUNEL, and caspase-3 activity assay was used to detect cell proliferation and apoptosis in THCA cells. Correlated genes of TNFRSF12A were identified using LinkedOmics database and subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Western blot analysis was performed to determine proliferating cell nuclear antigen (PCNA), cyclin D1 (CCND1), Bax, and Bcl-2 expression and to analyze the effect of TNFRSF12A on mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) pathways. Results showed that TNFRSF12A was increased in THCA tissue samples and cells. KEGG analysis showed that correlated genes of TNFRSF12A were significantly enriched in MAPK and NF-κB signaling pathways. Moreover, TNFRSF12A knockdown inactivated the MAPK and NF-κB signaling pathways in THCA cells. TNFRSF12A silencing alone or combined with inhibitor of ERK (PD98059), JNK (SP600125), p38 (SB203580), or NF-κB (Bay 11-7082) impeded cell proliferation and reduced PCNA and CCND1 expression in THCA cells. Meanwhile, TNFRSF12A knockdown alone or combined with PD98059, SP600125, SB203580, or Bay 11-7082 facilitated cell apoptosis, increased caspase-3 activity, downregulated Bcl-2 expression, and upregulated Bax expression in THCA cells. TNFRSF12A knockdown alone or combined with PD98059, SP600125, SB203580, or Bay 11-7082 also decreased the expression levels of proinflammatory cytokines IL-1β, IL-6, and IL-8 in THCA cells. On the contrary, TNFRSF12A overexpression showed an opposite effect. Treatment with PD98059, SP600125, SB203580, or Bay 11-7082 reversed the effects of TNFRSF12A overexpression on cell proliferation, apoptosis, and proinflammatory cytokine expression. In conclusion, the effects of TNFRSF12A on proliferation, apoptosis, and proinflammatory cytokine expression in THCA cells were regulated by the MAPK and NF-κB pathways.

Effect of tRF-28-ZJ47D112Z4DZ on the pathogenesis of gastric cancer through targeting cyclin D1/CDK4 and its early diagnostic value.

475 Background: Gastric cancer exhibits high incidence and mortality rates worldwide. Current research on gastric cancer aims to discover stable and effective early diagnostic markers, as well as uncover the disease's pathogenesis. tRNA-derived fragment (tRF) is a newly discovered regulatory non-coding RNA, which plays an important role in tumor diagnosis and treatment. Methods: This study, through transcriptome sequencing and MINTBase v2.0 database, screened out a new molecule tRF-28-ZJ47D112Z4DZ (tRF-28) related to gastric cancer. An absolute quantitative method was established to quantify the level of tRF-28 in plasma. ROC curve, survival curve, Cox regression model were constructed, and clinicopathological factors were collected to analyze the diagnostic value of tRF-28. "Overexpression" and "knockdown" tRF-28 gastric cancer cell models and xenograft tumor models were established to study the effects of tRF-28 on cell proliferation. Using bioinformatics, qRT-PCR, Western blot, dual luciferase reporter gene, fluorescence in situ hybridization, RNA binding protein immunoprecipitation assay, rescue experiment and immunohistochemistry to study the regulatory role of tRF-28 by binding to the 3'UTR of CCND1. Results: This study clarified the tRF-28 levels in the plasma of healthy individuals, and gastric benign and malignant lesions, providing a new candidate biomarker for the early diagnosis of gastric cancer. Through upregulating and downregulating the level of tRF-28 in gastric cancer cells, it was revealed that tRF-28 inhibits the proliferation of gastric cancer cells in vivo and in vitro by regulating key proteins such as Cyclin D1, CDK4, Rb, and p-Rb. In studying specific molecular mechanisms, tRF-28 and CCND1 were co-located in the cytoplasm. tRF-28 combines with Ago2 protein to form a complex and plays a role in silencing the target gene CCND1 in the cytoplasm. The rescue experiments revealed that silencing the target gene CCND1 can reverse the carcinogenesis effect of tRF-28 inhibitors, clarifying the biological functions of tRF-28 by regulating CCND1. Conclusions: Based on a series of research findings, we elucidated that tRF-28 form a complex with Ago2 protein, binding to the 3' UTR of CCND1 to downregulate Cyclin D1, CDK4, and other critical proteins, thereby inhibiting the proliferation of gastric cancer cells. Our study aims to offer new insights into identifying potential early diagnostic markers and therapeutic targets for gastric cancer.

Long non-coding RNA LINC01224 plays an oncogenic role in endometrial cancer via miR-4673/TPX2 axis and activating Wnt/β-catenin signaling pathway.

Endometrial cancer (EC) is a prevalent gynecological malignancy with a rising incidence and poor prognosis in advanced cases. Long non-coding RNAs (lncRNAs) have been implicated in various cancers, including EC. This study explores the role of lncRNA Linc01224 in EC. Analyzing TCGA data, we found Linc01224 expression significantly elevated in EC tissues, correlating with poor prognosis. Clinical samples validated these findings, showing higher Linc01224 levels in tumor tissues. Knockdown of Linc01224 in EC cell lines (Hec-1-B and Ishikawa) inhibited proliferation, migration, and promoted apoptosis, alongside increased Bax and decreased BCL2 expression. Furthermore, Linc01224 knockdown notably reduced Wnt2/β-catenin pathway activation. We identified TPX2 as a target of miR-4673, which is regulated by Linc01224 through a competing endogenous RNA (ceRNA) mechanism. Dual-luciferase reporter assays confirmed miR-4673 binding to Linc01224 and TPX2. Rescue experiments revealed that TPX2 knockdown reversed Linc01224-induced proliferation and migration, highlighting TPX2's pivotal role in Linc01224's oncogenic function. In vivo, Linc01224 knockdown significantly impeded tumor growth and metastasis in a xenograft model, with decreased expression of c-Myc, Cyclin D1, and β-catenin. These findings reveal a novel ceRNA regulatory axis involving Linc01224, miR-4673, and TPX2, elucidating Linc01224's role in EC progression through the Wnt2/β-catenin pathway. Linc01224 emerges as a potential biomarker and therapeutic target for EC prognosis and treatment.

Anticancer and therapeutic efficacy of XPO1 inhibition in pancreatic ductal adenocarcinoma through DNA damage and modulation of miR-193b/KRAS/LAMC2/ERK/AKT signaling cascade.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and grave malignancies with confined and ineffective therapeutic options. XPO1 is a critical regulator of nuclear export and activation of tumor suppressor proteins. The present study evaluated the therapeutic potential and molecular mechanisms of XPO1 inhibition against PDAC. Firstly, we observed significant overexpression of XPO1 transcript in 179 PDAC patients than 171 normal pancreatic tissues in TCGA transcriptomic dataset. Higher XPO1 transcript levels displayed worse overall and disease-free survival. Further, we confirmed significant upregulation of XPO1 in a panel of PDAC cells. Eltanexor treatment resulted in significant inhibition of cell viability, clonogenic growth, migration, and epithelial-mesenchymal transition (EMT), along with the induction of cell cycle arrest. Mechanistically, eltanexor modulated the expression of key proteins including p21, p27, p53, cyclin B1, cyclin D1, c-Myc, N-cadherin, vimentin, E-cadherin associated with the cell viability, growth, cell cycle and EMT. Additionally, the eltanexor treatment resulted in marked increase in expression of γH2AX, and cleaved PARP, cleaved caspase-9 leading to induction of DNA damage and apoptosis of PDAC cells, respectively. Moreover, eltanexor treatment regulated the expression of key non-coding RNAs including miR193b, DINO, MALAT-1, H19, and SOX21-AS1 linked with tumorigenesis. Our results revealed a correlation among miR193b/KRAS/LAMC2, XPO1/KRAS, and LAMC2/KRAS. The findings also revealed that eltanexor treatment rescued the expression of miR193b which acts as a sponge for LAMC2 and KRAS resulting in the suppression of AKT/ERK downstream signaling cascade in PDAC. Interestingly, the combination of eltanexor with gemcitabine showed significant anticancer activity in PDAC cells. Altogether, our findings revealed the crucial role of XPO1 in modulating the expression of oncogenic proteins, ncRNAs, and DNA damage during PDAC progression as well as identified novel therapeutic miR-193b/KRAS/LAMC2/ERK/AKT axis.

CCN2 functions as a modulator of cell cycle regulation in human dermal fibroblasts.

CCN2 is widely regarded as a profibrotic factor involved in fibrotic disorders by regulating extracellular matrix (ECM). We report here that CCN2 functions as a critical cell cycle regulator in primary human dermal fibroblasts (HDFs). siRNA-mediated knockdown of CCN2 halted proliferation of primary HDFs, which was rescued by a siRNA-resistant CCN2 expression vector. Furthermore, CCN2 knockdown caused a significant accumulation of cells in G1/G0 phase and blocked entry into S-phase. Mechanistically, CCN2 knockdown blocked cyclin E and CDK4/cyclin D nuclear translocation, and abrogated CDK2 activity. Markedly, CCN2 translocated to the nucleus and co-localized with cyclin D1 upon cell cycle stimulation. Finally, we show that CCN2, a bona fide YAP/TAZ target gene, partially mediates YAP/TAZ-dependent proliferation of primary HDFs. These data provide evidence of a novel CCN2 function as a cell cycle regulator in primary HDFs proliferation, in addition to its known role in ECM regulation.

Cyclin E Expression and p16 Loss Are Strong Prognostic Biomarkers in Primary Invasive Cutaneous Melanoma.

The traditional melanoma staging system is not ideal for predicting patients' individual risk of disease recurrence and death. Subsequently, suboptimal adjuvant treatment may be offered. Hence, identification of biomarkers to optimize risk stratification is warranted. Cyclins and cyclin-dependent kinase inhibitors, key players in the cell cycle regulation, are candidate prognostic biomarkers. Herein, their expression and prognostic value in melanoma are studied. The expression of Cyclin D1, Cyclin E, p16, p21, and p27 were assessed using immunohistochemistry in samples from 59 patients with melanoma and correlated with clinicopathological parameters, as well as relapse-free survival (RFS) and overall survival (OS). Cyclin E expression in the nucleus, observed in 19% of patients, was correlated with Breslow thickness (p=0.017), whereas p16 loss in the cytoplasm and nucleus, detected in 68% and 61% of patients respectively, was correlated with Breslow thickness (p=0.001) and ulceration (p=0.035). The high expression of Cyclin E in the nucleus (p<0.001) and the loss of p16 in the cytoplasm (p=0.012) and nucleus (p=0.002) were associated with shorter OS. Cyclin E in the nucleus was a prognostic factor for RFS and OS at least as strong as Breslow thickness. Expression of Cyclin E and loss of p16 appeared to be significant prognostic biomarkers, with Cyclin E being prognostically at least as strong as Breslow thickness regarding RFS and OS. Both biomarkers may potentially be used to improve stratification of individual patient's risk of recurrent disease and survival, and subsequently optimize adjuvant treatment.

Design, synthesis, and structure-activity relationships of five-membered heterocyclic incorporated aryl(alkyl)azoles: From antiproliferative thiazoles to safer anticonvulsant oxadiazoles.

In the current study, a novel series of 1,2,4-oxadiazoles were designed, synthesized, and evaluated for their biological activities. A cell-based antiproliferative screening was accomplished on the newly synthesized 1,2,4-oxadiazoles along with our previously reported aryl(alkyl)azoles (AAAs) containing middle heterocyclic cores thiazole and oxazole. Among the tested compounds, naphthyl- thiazoles demonstrated higher antiproliferative activity and B3 was identified as the most potent compound with IC50 values in the range of 2.03-3.6µM against SH-SY5Y neuroblastoma, HT-29 colorectal adenocarcinoma, and fibroblast cells (ten folds more potent than 5-FU and irinotecan). Further investigation revealed that B3 strongly inhibits tubulin polymerization with an IC50 of 0.79µM, outperforming the reference drug colchicine (IC50=1.46µM). In addition, evaluation of B3 on the expression level of BAX, BCL2, and CYCLIN D1 genes indicated the suppression of the cell cycle in the genome level. Interestingly, the 1,2,4-oxadiazole congeners displayed optimal anticonvulsant activity with significantly reduced cytotoxicity. Among the oxadiazole series, compound D4 featuring a 1,2,4-triazole head group demonstrated the highest activity in the maximal electroshock (MES) and pentylenetetrazol (PTZ) tests, with ED50 values of 2.23 and 24.60mg/kg, respectively. In vivo evaluations suggested that D4 exerts its anticonvulsant effects by enhancing GABAA currents. In conclusion, our findings indicated that B3 in the thiazole congeners is a promising drug candidate for cancer treatment with a well-defined mechanism of action. Moreover, D4 and its congeners containing oxadiazole core are much safer anti-seizures which have potential for preclinical considerations as novel anticonvulsants.

Exploring the antiproliferative effect of PI3K/Akt/mTOR pathway and CDK4/6 inhibitors in human papillomavirus‑positive and ‑negative head and neck squamous cell carcinoma cell lines.

Human papillomavirus (HPV)‑positive and -negative head and neck squamous cell carcinoma (HNSCC) are often associated with activation of the phosphatidylinositol 3‑kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway due to mutations or amplifications in PI3KCA, loss of PTEN or activation of receptor tyrosine kinases. In HPV‑negative tumors, CDKN2A (encoding p16 protein) inactivation or CCND1 (encoding Cyclin D1 protein) amplification frequently results in sustained cyclin‑dependent kinase (CDK) 4/6 activation. The present study aimed to investigate the efficacy of the CDK4/6 inhibitors (CDKi) palbociclib and ribociclib, and the PI3K/Akt/mTOR pathway inhibitors (PI3Ki) gedatolisib, buparlisib and alpelisib, in suppressing cell viability of HPV‑positive and ‑negative HNSCC cell lines. Inhibitor efficacy was assessed in vitro using MTT assay and western blotting analysis. Cell cycle analysis was performed using flow cytometry and apoptosis was assessed using annexin V staining. Metabolic changes in terms of glycolysis and oxidative metabolism were measured by Seahorse XF96 extracellular Flux analysis. The results of the present study showed that both HPV‑positive and ‑negative HNSCC cell lines were sensitive to PI3Ki. In general, PI3Ki decreased PI3K/Akt/mTOR pathway activity, resulting in apoptosis, and decreased oxidative and glycolytic metabolism. The CDKi were particularly effective in blocking HPV‑negative cell line viability, showing decreased retinoblastoma expression and G1‑phase cell cycle arrest, whereas apoptosis was not induced. Thus, PI3Ki and CDKi efficiently inhibited their respective pathways and HNSCC cell viability in vitro, with the latter occurring only in HPV‑negative cell lines. Whereas PI3Ki induced apoptosis and attenuated cellular metabolism, CDKi led to cell cycle arrest. Further research should be performed to elucidate whether (a combination of) these inhibitors may be effective therapeutic agents for patients with HNSCC.

Gamma-tocotrienol Inhibits Proliferation and Growth of HSD17B4 Overexpressing HepG2 Liver Cancer Cells.

Hydroxysteroid 17-beta dehydrogenase 4 (HSD17B4) is involved in the progression of hepatocellular carcinoma (HCC). This study aimed to investigate the inhibitory effect of gamma-tocotrienol (γ-T3) on the proliferation and growth of HSD17B4-overexpressing HepG2 cells. HepG2 cells were transfected with empty or HSD17B4-overexpressing plasmids, followed by vitamin E (VE) or γ-T3 treatment. MTS assay, Western blotting, qRT-PCR, and flow cytometry were employed to assess cell proliferation, protein expression, mRNA levels, and apoptosis. HSD17B4 interaction with γ-T3 was assessed by quantifying γ-T3 in the collected precipitate of HSD17B4 using anti-flag magnetic beads. Tumor xenografts were established in NSG mice, and tumor growth was monitored. HSD17B4 overexpression significantly promoted HepG2 cell proliferation, which was effectively counteracted by VE or γ-T3 treatment in a dose-dependent manner. VE and γ-T3 did not exert their effects through direct regulation of HSD17B4 expression. Instead, γ-T3 was found to interact with HSD17B4, inhibiting its activity in catalyzing the conversion of estradiol (E2) into estrone. Moreover, γ-T3 treatment led to a reduction in cyclin D1 expression and suppressed key proliferation signaling pathways, such as ERK, MEK, AKT, and STAT3. Additionally, γ-T3 promoted apoptosis in HSD17B4-overexpressing HepG2 cells. In an in vivo model, γ-T3 effectively reduced the growth of HepG2 xenograft tumors. In conclusion, our study demonstrates that γ-T3 exhibits potent anti-proliferative and anti-tumor effects against HepG2 cells overexpressing HSD17B4. These findings highlight the therapeutic potential of γ-T3 in HCC treatment and suggest its role in targeting HSD17B4-associated pathways to inhibit tumor growth and enhance apoptosis.

3,6-Anhydro-L-galactose suppresses mouse lymphocyte proliferation by attenuating JAK-STAT growth factor signal transduction and G1-S cell cycle progression.

Recombinant GH16B β-agarase-catalyzed liquefaction of 5-7%(w/v) melted agarose at 50°C completely hydrolyzed agarose into neoagarohexaose (NA6) and neoagarotetraose (NA4). Subsequent saccharification by recombinant GH50A β-agarase or recombinant GH50A β-agarase/recombinant GH117A α-neoagarobiose hydrolase at 35°C converted NA6/NA4 into neoagarobiose (NA2) or 3,6-anhydro-L-galactose (L-AHG)/D-galactose, respectively. Purification of NA6/NA4 and NA2 was achieved by Sephadex G-15 column chromatography, while L-AHG was purified by Sephadex G-10, achieving≥98% purity. L-AHG (25-200μg/mL), but not NA2, NA4, or NA6, inhibited the proliferation of immobilized anti-CD3/anti-CD28-activated T cells and immobilized anti-CD40+soluble anti-IgM+interleukin (IL)-4-activated B cells. This inhibition impacted the G1-S traverse in the cell cycle without influencing CD69 expression and p27Kip1 down-regulation, markers of the exit from G0 into G1 phase in activated lymphocytes. L-AHG impeded cyclin-dependent kinases (CDKs)-driven retinoblastoma phosphorylation, necessary for the G1-S traverse, by reducing the activating phosphorylation of CDKs (CDK4, CDK2, and CDK1) and lowering cyclin D3, cyclin A2 and cyclin B1 levels. Furthermore, L-AHG diminished the production of growth factors, including IL-2 in activated T cells and IL-6 in activated B cells. The antiproliferative effect of L-AHG on T cells was partially restored by exogenous IL-2 but was unaffected by exogenous IL-6 on B cells. L-AHG inhibited the activating phosphorylation of Janus kinase 1 (JAK1), affecting signal transducer and activator of transcription 1 (STAT1) and STAT3 signaling. These results demonstrate that L-AHG may serve as a novel immunosuppressant by impairing JAK-STAT growth factor signaling and G1-S cell cycle progression in T and B lymphocytes.

Synergistic effects of doxorubicin and coumarin on colorectal cancer cell proliferation and EMT modulation: in silico and in vitro study

Background Colorectal cancer (CRC) ranks as the third most common cancer and the second leading cause of cancer-related deaths globally. Doxorubicin (DOX), an anti-metabolite and anti-cancer agent, has shown promise in CRC treatment, while coumarin, a natural antioxidant, may help slow CRC growth. Although the combination of coumarin and DOX has been studied in other cancer types, its effectiveness in CRC remains unexamined. This study explores the impact of DOX and coumarin, individually and combined, on CRC cell growth and death, specifically focusing on C26 cells in vitro. Methods The MTT assay was used to assess cell proliferation, and RT-qPCR was employed to measure the expression of β-catenin, Twist, Cyclin D1, and ZEB-1. Results Both DOX and coumarin independently inhibited C26 cell growth, and their combination had a synergistic effect. RT-qPCR analysis indicated a reduction in Twist, Cyclin D1, and ZEB-1 expression following combination treatment, suggesting these molecules are involved in the observed anti-cancer effects. Molecular docking revealed that coumarin can target Twist, Cyclin D1, and ZEB-1 at the protein level. Conclusion Our findings validate the synergistic effect of DOX and coumarin in inhibiting the proliferation of C26 cells and propose the involvement of the Wnt pathway as a downstream mediator in this treatment. Future research is required to confirm the clinical applicability of this combined treatment for CRC and to elucidate its mechanisms within the body.