Hydrangea serrata extract exerts tumor inhibitory activity against hepatocellular carcinoma HepG2 cells via inducing p27/CDK2-mediated cell cycle arrest and apoptosis

Objective:To examine the inhibitory effect of Hydrangea serrata extract against hepatocellular carcinoma HepG2 cells and its underlying mechanisms.Methods:The effects of Hydrangea serrata extract on growth inhibition of tumor cells and spheroids were assessed using MTT and 3D culture assays.Quantitative real-time PCR and Western blot analyses were employed to investigate the changes in mRNA and protein expression levels of molecules related to cell cycle and apoptosis.Results:Hydrangea serrata extract effectively inhibited the growth of both tumor cells and spheroids.The extract also significantly upregulated p27 mRNA expression and downregulated CDK2 mRNA expression,leading to cell cycle arrest.Moreover,increased BAX/Bcl-2 ratio as well as caspase-9 and-3 were observed after treatment with Hydrangea serrata extract,indicating the induction of tumor cell apoptosis.Conclusions:Hydrangea serrata extract has the potential to alleviate tumors by effectively modulating cell-cycle-related gene expressions and inducing apoptosis,thereby inhibiting tumor growth.

Antitumor Effect of Apcin on Endometrial Carcinoma via p21-Mediated Cell Cycle Arrest and Apoptosis

Objective Endometrial carcinoma(EC)is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates.This underscores the critical need for novel therapeutic targets.One such potential target is cell division cycle 20(CDC20),which has been implicated in oncogenesis.This study investigated the effect of the CDC20 inhibitor Apcin on EC and elucidated the underlying mechanism involved.Methods The effects of Apcin on EC cell proliferation,apoptosis,and the cell cycle were evaluated using CCK8 assays and flow cytometry.RNA sequencing(RNA-seq)was subsequently conducted to explore the underlying molecular mechanism,and Western blotting and coimmunoprecipitation were subsequently performed to validate the results.Animal studies were performed to evaluate the antitumor effects in vivo.Bioinformatics analysis was also conducted to identify CDC20 as a potential therapeutic target in EC.Results Treatment with Apcin inhibited proliferation and induced apoptosis in EC cells,resulting in cell cycle arrest.Pathways associated with apoptosis and the cell cycle were activated following treatment with Apcin.Notably,Apcin treatment led to the upregulation of the cell cycle regulator p21,which was verified to interact with CDC20 and consequently decrease the expression of downstream cyclins in EC cells.In vivo experiments confirmed that Apcin treatment significantly impeded tumor growth.Higher CDC20 expression was observed in EC tissue than in nonmalignant tissue,and increased CDC20 expression in EC patients was associated with shorter overall survival and progress free interval.Conclusion CDC20 is a novel molecular target in EC,and Apcin could be developed as a candidate antitumor drug for EC treatment.

NCAPD2 serves as a potential prognostic biomarker for lung adenocarcinoma and promotes cell proliferation,migration,invasion and cell cycle in vitro

Objectives:The pro-oncogenic effects of NCAPD2 have been extensively studied across various tumor types;however,its precise role within the context of lung adenocarcinoma(LUAD)remains elusive.This study aims to elucidate the biological functions of NCAPD2 in LUAD and unravel the underlying mechanistic pathways.Methods:Utilizing bioinformatics methodologies,we explored the differential expression of NCAPD2 between normal and tumor samples,along with its correlations with clinical-pathological characteristics,survival prognosis,and immune infiltration.Results:In the TCGA-LUAD dataset,tumor samples demonstrated significantly elevated levels of NCAPD2 expression compared to normal samples(p<0.001).Clinically,higher NCAPD2 expression was notably associated with advanced T,N,and M stages,pathologic stage,gender,smoking status,and diminished overall survival(OS).Moreover,differentially expressed genes(DEGs)associated with NCAPD2 were predominantly enriched in pathways related to cell division.Immune infiltration analysis revealed that NCAPD2 expression levels were linked to the infiltration of memory B cells,naïve CD4+T cells,activated memory CD4+T cells,and M1 macrophages.In vitro experiments demonstrated that silencing NCAPD2 suppressed LUAD cell proliferation,migration,invasion,epithelial-mesenchymal transition(EMT),and cell cycle progression.Conclusions:In summary,NCAPD2 may represent a promising prognostic biomarker and novel therapeutic target for LUAD.

基于CycleGAN的虚拟定量差分相衬成像用于红细胞分类

目的通过深度学习技术实现虚拟定量差分相衬(Virtual Quantitative Differential Phase Contrast,V-qDPC)重建,提高定量相位成像衬度和鲁棒性,为无标记红细胞的全自动分类提供新思路。方法通过对LED照明进行编码获得明场图像和差分相衬图像,通过相位重建可获得定量差分相衬(Quantitative Differential Phase Contrast,qDPC)图像;采用循环一致生成对抗网络(Cycle-consistent Generative Adversarial Network,CycleGAN)完成明场图像到qDPC图像的端到端映射。结果基于CycleGAN生成的V-qDPC图像,实验参数当λ=7和β=0.5时,V-qDPC图像质量最优;相比光学重建的qDPC图像有更好的鲁棒性和抗噪声能力;使用AlexNet、ResNet50和VggNet三种网络模型比较无标记红细胞形态的自动分类,结果表明V-qDPC图像比qDPC图像具有更好分类性能。结论与传统的基于多幅倾斜照明图像的qDPC重建相比,V-qDPC算法具有更好的相位图像质量和鲁棒性,适合以高精度和高效率实现全自动细胞分类,同时省去了成像光路和硬件支持,有望应用于生物医学研究。

Cdk5 and aberrant cell cycle activation at the core of neurodegeneration

Neurodegenerative diseases are caused by the progressive loss of specific neurons.The exact mechanisms of action of these diseases are unknown,and many studies have focused on pathways related to abnormal accumulation and processing of proteins,mitochondrial dysfunction,and oxidative stress leading to apoptotic death.However,a growing body of evidence indicates that aberrant cell cycle re-entry plays a major role in the pathogenesis of neurodegeneration.The activation of the cell cycle in mature neurons could be promoted by several signaling mechanisms,including c-Jun N-terminal kinases,p38 mitogen-activated protein kinases,and mitogen-activated protein kinase/extracellular signal-regulated kinase cascades;post-translational modifications such as Tau-phosphorylation;and DNA damage response.In all these events,implicated Cdk5,a proline-directed serine/threonine protein kinase,seems to be responsible for several cellular processes in neurons including axon growth,neurotransmission,synaptic plasticity,neuronal migration,and maintenance of neuronal survival.However,under pathological conditions,Cdk5 dysregulation may lead to cell cycle re-entry in post-mitotic neurons.Thus,Cdk5 hyperactivation,by its physiologic activator p25,hyper-phosphorylates downstream substrates related to neurodegenerative diseases.This review summarizes factors such as oxidative stress,DNA damage response,signaling pathway disturbance,and Ubiquitin proteasome malfunction contributing to cell cycle re-entry in post-mitotic neurons.It also describes how all these factors are linked to a greater or lesser extent with Cdk5.Thus,it offers a global vision of the function of cell cycle-related proteins in mature neurons with a focus on Cdk5 and how this protein contributes to the development of Alzheimer’s disease,Parkinson’s disease,amyotrophic lateral sclerosis,and Huntington’s disease by cell cycle activation.

Wild pink bayberry free phenolic extract induces mitochondria-dependent apoptosis and G0/G1 cell cycle arrest through p38/MAPK and PI3K/Akt pathway in MDA-MB-231 cancer cells

Polyphenol-rich foods have been shown to be good for cancer prevention as powerful antioxidants. In this study, the mechanisms of wild pink bayberry free phenolic extract(WPBFE)inhibiting the proliferation and inducing apoptotic of MDA-MB-231 breast cancer cells was examined. The main phenolic acids and flavonols in WPBFE were gallic acid((18.83 ± 0.44)μg/g FW)and myricetin((1.52 ± 0.05)μg/g FW), respectively. The maximum inhibition rate of WPBFE at non-cytotoxicity dose(below 80 mg/mL)was 81%. Western blotting analysis showed that WPBFE could cause the arrest of cell cycle in G0/G1 phase by down-regulating expression levels of PCNA, CDK4, cyclin D1 and up-regulating the expression level of p21. Meanwhile, WPBFE induced apoptosis through initiating the mitochondrial death pathway by up-regulating cleaved caspase-3 and enhancing the ratio of Bax/Bcl-2, with the maximum expression levels of 1.29 and 2.03 folds that of control group, respectively. Further study of the upstream protein, we found that WPBFE down-regulated TRAF2, while upregulated p-ASK1, p-p38 and p-p53. Furthermore, WPBFE could down-regulate the expression of p-PI3K and p-Akt. These observations indicated that WPBFE might play an anticancer role through regulating the p38 MAPK together with PI3K/Akt pathway.

Combinatorial effect of diclofenac with piperine and D-limonene on inducing apoptosis and cell cycle arrest of breast cancer cells

Objective:To investigate the potential synergistic activity of diclofenac with piperine and D-limonene in inducing apoptosis and cell cycle arrest in breast cancer MCF-7 cells.Methods:Molecular docking study was conducted to evaluate the binding affinity of diclofenac with piperine and D-limonene against p53,Bax,and Bcl-2.The MTT assay was used to determine IC50,and the Chou-Talay method was used to determine the synergistic concentration of the combination treatment of diclofenac plus piperine and diclofenac plus D-limonene.Apoptosis detection,cell cycle arrest,reactive oxygen species production,and mitochondrial membrane potential were also investigated.Results:Diclofenac,piperine,and D-limonene showed potent binding affinity for p53,Bax,and Bcl-2.Diclofenac plus piperine and diclofenac plus D-limonene enhanced the formation of reactive oxygen species,which also had an effect on the mitochondrial membrane’s integrity and caused DNA fragmentation.Diclofenac plus piperine and diclofenac plus D-limonene arrested the cells in the sub-G0phase while drastically lowering the percentage of cells in the G2/M phase.Furthermore,the elevated apoptosis in the combined therapy was confirmed by annexin V/propidium iodide staining.Conclusions:The combined therapy prominently enhanced the antiproliferative and apoptotic effects on MCF-7 cells compared with treatment with diclofenac,piperine,and D-limonene alone.

The role of FZR1 in tumorigenesis: Focus on cell-cycle control

Fizzy-related protein homolog 1 (FZR1) mainly functions as a specific activator of the anaphase-promotingcomplex/cyclosome (APC/C) in the cell cycle and controls the G0 and G1 phases of the cell cycle. We highlightrecent work that has studied the role of FZR1 in tumorigenesis, growth, differentiation, and genome stability throughcell-cycle control. We summarize the current state of knowledge regarding FZR1 structure, function, and the distinctways of APC/C dysregulation in solid tumors and hematologic malignancies. We also discuss novel approaches fortargeting the FZR1 as a cancer therapy and research area for future work.

Expression dynamics of periodic transcripts during cancer cell cycle progression and their correlation with anticancer drug sensitivity

Background:The cell cycle is at the center of cellular activities and is orchestrated by complex regulatory mechanisms,among which transcriptional regulation is one of the most important components.Alternative splicing dramatically expands the regulatory network by producing transcript isoforms of genes to exquisitely control the cell cycle.However,the patterns of transcript isoform expression in the cell cycle are unclear.Therapies targeting cell cycle checkpoints are commonly used as anticancer therapies,but none of them have been designed or evaluated at the alternative splicing transcript level.The utility of these transcripts as markers of cell cycle-related drug sensitivity is still unknown,and studies on the expression patterns of cell cycle-targeting drug-related transcripts are also rare.Methods:To explore alternative splicing patterns during cell cycle progression,we performed sequential transcriptomic assays following cell cycle synchronization in colon cancer HCT116 and breast cancer MDA-MB-231 cell lines,using flow cytometry and reference cell cycle transcripts to confirm the cell cycle phases of samples,and we developed a new algorithm to describe the periodic patterns of transcripts fluctuating during the cell cycle.Genomics of Drug Sensitivity in Cancer(GDSC)drug sensitivity datasets and Cancer Cell Line Encyclopedia(CCLE)transcript datasets were used to assess the correlation of genes and their transcript isoforms with drug sensitivity.We identified transcripts associated with typical drugs targeting cell cycle by determining correlation coefficients.Cytotoxicity assays were used to confirm the effect of ENST00000257904 against cyclin dependent kinase 4/6(CDK4/6)inhibitors.Finally,alternative splicing transcripts associated with mitotic(M)phase arrest were analyzed using an RNA synthesis inhibition assay and transcriptome analysis.Results:We established high-resolution transcriptome datasets of synchronized cell cycle samples from colon cancer HCT116 and breast cancer MDA-MB-231 cells.The results of the cell cycle assessment showed that 43,326,41,578 and 29,244 transcripts were found to be periodically expressed in HeLa,HCT116 and MDA-MB-231 cells,respectively,among which 1280 transcripts showed this expression pattern in all three cancer cell lines.Drug sensitivity assessments showed that a large number of these transcripts displayed a higher correlation with drug sensitivity than their corresponding genes.Cell cycle-related drug screening showed that the level of the CDK4 transcript ENST00000547281 was more significantly associated with the resistance of cells to CDK4/6 inhibitors than the level of the CDK4 reference transcript ENST00000257904.The transcriptional inhibition assay following M phase arrest further confirmed the M-phase-specific expression of the splicing transcripts.Combined with the cell cycle-related drug screening,the results also showed that a set of periodic transcripts,for example,ENST00000314392(a dolichylphosphate mannosyltransferase polypeptide 2 isoform transcript),was more associated with drug sensitivity than the levels of their corresponding gene transcripts.Conclusions:In summary,we identified a panel of cell cycle-related periodic transcripts and found that the levels of transcripts of drug target genes showed different values for predicting drug sensitivity,providing novel insights into alternative splicing-related drug development and evaluation.

Novel coumarone-derived(S,E)-4-(4-fluorobenzylidene)-3-phenylchroman-3-ol inhibits muscle-invasive bladder cancer cells by repressing the S and G2 cell cycle phases

Background:This study aimed to select compounds with unique inhibitory effects on muscle-invasive bladder cancer(MIBC)from coumarone derivatives with similar parent nuclear structures and to reveal their tumor-suppressive effects using various approaches.Methods:Bladder cancer cell lines SW780 and T24,as well as human normal bladder epithelial cell line SV-HUC-1 were selected as the study model,and these urinary system cells were co-incubated with various concentrations of(S,E)-4-(4-methylbenzylidene)-3-phenylchroman-3-ol,(S,E)-4-(4-isocyanobenzylidene)-3-phenylchroman-3-ol,(S,E)-4-(4-fluorobenzylidene)-3-phenylchroman-3-ol(FPO),and(S,E)-3-phenyl-4-(4-(trifluoromethoxy)benzylidene)chroman-3-ol.Cell activity was detected using cell counting kit-8.FPO showed the strongest inhibitory effect on MIBC cells;therefore,it was selected for further experiments.We monitored the FPO-induced T24 cell morphological changes with an inverted microscope.The FPO-inhibited migration of T24 cells was examined using a cell scratch assay.We detected the clonogenic ability of T24 cells through a clone formation test and evaluated their proliferative ability using a 5-ethynyl-2’-deoxyuridine fluorescence staining kit.The inhibitory effect of FPO against the cell cycle was monitored using flow cytometry,and its suppressive effect on the DNA replication ability of T24 cells was detected using double fluorescence staining(Ki67 and phalloidin).Results:Among the four candidate coumarone derivatives,FPO showed the most significant inhibitory effect on MIBC cells and was less toxic to normal urothelial cells.FPO inhibited T24 cell growth in time and dose-dependent manners(the half-inhibitory concentration is 8μM).FPO significantly repressed the proliferation,migration,and clonogenic ability of bladder cancer T24 cells.Cell mobility was significantly inhibited by FPO:30μM FPO almost completely repressed migration occurred at after 24 h treatment.Moreover,FPO significantly suppressed the clonogenicity of bladder cancer cells in a dose-dependent manner.Mechanistically,FPO targeted the cell cycle,arresting the S and G2 phases on bladder cancer T24 cells.Conclusion:We discovered a novel anticancer chemical,FPO,and proposed a potential mechanism,through which it suppresses MIBC T24 cells by repressing the cell cycle in the S and G2 phases.This study contributes to the development of novel anticancer drugs for MIBC.

温胆汤含药血清对10 mmol/L谷氨酸环境下星型胶质细胞PI3K、Akt、mTOR表达的影响

目的 探讨温胆汤含药血清对10 mmol/L谷氨酸诱导的大鼠星型胶质细胞凋亡、周期及磷脂酰肌醇3-激酶(phosphoinositide 3-kinase, PI3K)、蛋白激酶B(protein kinase B,Akt)、哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)表达的影响。方法 将60只SPF级SD雄性大鼠随机分为5组,其中正常组20只,氯氮平组和温胆汤高、中、低剂量组各10只。正常组予20 g/kg 0.9%氯化钠溶液灌胃,氯氮平组予氯氮平原药20 mg/kg灌胃,温胆汤高、中、低剂量组分别予40、20、10 g/kg温胆汤灌胃,1次/d,共8 d。处死大鼠后取血,离心取血清,灭活除菌,EP管分装备用。将大鼠星型胶质细胞分为正常血清组、模型血清组,氯氮平含药血清组和温胆汤高、中、低剂量含药血清组,除正常血清组外,其余各组予10μmol/mL谷氨酸处理48 h后,流式细胞仪检测细胞凋亡和细胞周期;Western bloting、Real-time PCR分别测定细胞PI3K、Akt、mTOR蛋白和mRNA表达。结果 温胆汤含药血清可明显降低谷氨酸环境下大鼠星型胶质细胞凋亡率(P<0.01)和G0/G1期细胞占比(P<0.05),升高S期及G2/M期细胞占比;降低细胞P-P13K/P13K、P-ATK/AKT、P-mTOR/mTOR蛋白表达比值(温胆汤低剂量血清组除外,P<0.05)和细胞PI3K、Akt、mTOR mRNA表达(温胆汤低剂量血清组除外,P<0.01)。结论 温胆汤含药血清可有效调节大鼠星形胶质细胞PI3K、Akt、mTOR表达,达到保护神经细胞的目的,这可能是温胆汤治疗精神分裂症的作用机制之一。

青藤碱可有效抑制白细胞介素1β介导的髓核细胞凋亡

背景:椎间盘退变是导致脊柱退行性疾病的基础,然而目前尚无有效的治疗药物。目的:探讨青藤碱是否可以抑制白细胞介素1β诱导的髓核细胞凋亡及其分子机制。方法:采用胰酶联合Ⅱ型胶原酶消化法体外培养大鼠髓核细胞,并绘制细胞生长曲线,采用CCK-8法筛选合适的青藤碱药物浓度。将髓核细胞分为对照组、青藤碱组、白细胞介素1β组、青藤碱+白细胞介素1β组、锌原卟啉(血红素氧合酶1抑制剂)组、锌原卟啉+青藤碱组、锌原卟啉+白细胞介素1β组、青藤碱+锌原卟啉+白细胞介素1β组。分别检测各组髓核细胞增殖活性、活性氧含量、凋亡率及血红素氧合酶1的表达情况。结果与结论:①体外培养的大鼠髓核细胞呈现多角形、三角形、短楔形等形态,其呈现“S”型曲线生长,接种第1-3天生长缓慢,第4-6天生长迅速,第七八天生长速度缓慢,进入“平台期”,细胞数量不再增加;②当青藤碱的浓度≤80μmol/L时,髓核细胞的增殖活性不会受到显著影响(P>0.05);③白细胞介素1β可以显著降低髓核细胞的增殖活性,增加活性氧含量,导致细胞凋亡(P<0.01);④当采用青藤碱干预后,不仅可以促进血红素氧合酶1的表达(P<0.05),而且可以抑制白细胞介素1β诱导的髓核细胞增殖活性降低、活性氧含量和凋亡率增加(P<0.05),其作用可被锌原卟啉逆转(P<0.01)。

miR-567通过调控CDK8在NSCLC增殖、迁移和细胞周期中的作用及其临床相关性研究

目的探讨微小RNA(miR)-567通过调控周期蛋白依赖性激酶8(CDK8)在非小细胞肺癌(NSCLC)增殖、迁移和细胞周期中的作用及其临床相关性研究。方法收集40例NSCLC患者的肿瘤组织和临近癌旁组织,采用实时荧光定量PCR(qRT-PCR)检测miR-567和CDK8的表达。将miR-NC mimic、miR-567 mimic、oe-NC和oe-CDK8转染至A549和H1975细胞中,使用qRT-PCR检测miR-567和CDK8的表达,CCK-8法检测细胞增殖水平,Transwell法检测细胞迁移水平,流式细胞术检测细胞周期变化。通过荧光素酶报告基因实验检测miR-567与CDK8的靶向性。结果在NSCLC患者的肿瘤组织中,miR-567表达降低,而CDK8表达升高,二者呈负相关(P<0.05)。在A549和H1975细胞中,miR-567 mimic组相较于miR-NC mimic组,miR-567表达升高,CDK8表达降低,细胞增殖和迁移水平降低,细胞G1期比例升高,S期比例降低;miR-567 mimic组在正常型CDK8中,荧光强度低于miR-NC mimic组;miR-567 mimic+oe-CDK8组相较于miR-567 mimic+oe-NC组,CDK8表达升高,细胞增殖和迁移水平升高,细胞G1期比例降低,S期比例升高。结论miR-567通过靶向抑制CDK8表达,控制肿瘤细胞在S期阻滞,从而抑制NSCLC的增殖和迁移能力。

骨髓间充质干细胞来源外泌体调节大鼠肝细胞凋亡的机制

背景:骨髓间充质干细胞可释放大量外泌体,关于骨髓间充质干细胞来源外泌体对肝细胞凋亡的影响以及具体机制还没有完全阐明。目的:探索骨髓间充质干细胞来源外泌体所携带的miR-21-5p对大鼠肝脏细胞凋亡的影响及其作用机制。方法:分离大鼠骨髓间充质干细胞,将miR-21-5p NC或miR-21-5p inhibitor转染到骨髓间充质干细胞中,采用超速离心法提取外泌体,命名为(BMSCs+miR-21-5p NC)-Exos,(BMSCs+miR-21-5p inhibitor)-Exos,将外泌体与BRL大鼠肝细胞共培养,观察抑制miR-21-5p表达后对大鼠肝细胞凋亡的影响。通过双荧光素酶报告基因检测验证外泌体中miR-21-5p和PIK3R1之间的靶向关系;TUNEL检测外泌体中miR-21-5p直接靶向PIK3R1激活PI3K/AKT信号通路对BRL大鼠肝细胞凋亡的影响。结果与结论:①双荧光素酶报告系统证实,PI3KR1野生型载体与miR-21-5p mimics共转染293T细胞时的荧光素酶活性显著低于PI3KR1突变型载体共转染组,表明miR-21-5p可靶向结合PIK3R1;②TUNEL检测结果显示:相比于(BMSCs+miR-21-5p NC)-Exos组,(BMSCs+miR-21-5p inhibitor)-Exos处理后BRL肝细胞凋亡率显著增加;与(BMSCs+miR-21-5p NC)-Exos组相比,加入AKT抑制剂LY294002之后,细胞凋亡率显著增加;③结果提示:外泌体可能通过miR-21-5p直接靶向PIK3R1激活PI3K/AKT信号通路抑制BRL大鼠肝细胞凋亡。

中药单体及复方在膝骨关节炎中的治疗进展

背景:膝骨关节炎是一种多因素导致的退行性疾病,其发病机制复杂,目前仍不清晰。中药在治疗膝骨关节炎方面富有成效,深入研究中药在膝骨关节炎中的治疗作用机制具有重要意义。目的:综述中药单体及复方在膝骨关节炎中的治疗进展,为有效防治膝骨关节炎提供思路和借鉴。方法:查阅国内外数据库从数据库建立至2022年发表的相关文献,设置中文检索词为“膝骨关节炎”“软骨破坏”“中药”“中药复方”“治疗”等,英文检索词为“Knee osteoarthritis”“Cartilage damage”“Traditional Chinese Medicine”“Chinese herbal compound”“Treatment”等,检索中国知网、万方、维普、PubMed、MEDLINE、Nature及Cochrane数据库,排除重复及陈旧无参考意义的文献,通过纳入和排除标准共纳入62篇标准文献进行探讨。结果与结论:①膝骨关节炎部分发病机制主要有免疫炎症反应、软骨细胞自噬与凋亡、血管内皮生长因子水平影响和生物力学失衡等;②中药治疗膝骨关节炎的作用机制主要集中在调节炎症因子水平、软骨细胞自噬与凋亡、血管内皮生长因子水平和改善软骨性能方面,以此延缓膝骨关节炎发生发展。

软骨细胞中生物钟基因Bmal1对细胞周期相关基因表达的影响

目的 探讨生物钟和细胞周期在骨关节炎(OA)软骨细胞中内在的关系,主要是时钟基因Bmal1对细胞周期相关基因的调控。方法 将胰岛素-转铁蛋白-硒(ITS)诱导后的软骨样ATDC5细胞分为normal组、OA组、LV-Bmal1组。采用CCK8法检测各组细胞活力;RT-qPCR法检测Bmal1、Per1、Wee1、Cdk1、Ccnb1和Mmp13 mRNA在各组中的表达;Western blot检测BMAL1、PER1、WEE1、CDK1、CCNB1和MMP13蛋白在各组中的表达水平;流式细胞测量术分析Bmal1对细胞周期中不同分期及细胞凋亡的影响;分析Bmal1对Per1、Wee1、Cdk1、Ccnb1和Mmp13的调控和它们在OA中发挥的作用。结果 与normal组相比,OA组细胞活力提高,Bmal1、Wee1的mRNA相对表达量下降,Per1、Cdk1、Ccnb1、Mmp13的mRNA相对表达量明显增加;LV-Bmal1组细胞活力下降,Bmal1、Wee1的mRNA相对表达量上升,Per1、Cdk1、Ccnb1、Mmp13的mRNA相对表达量下降(P<0.05);相关性分析显示,Bmal1与Wee1呈正相关,二者与Per1、Cdk1、Ccnb1或Mmp13存在负相关;Western blot结果显示,不同组别中蛋白表达水平的结果与PCR趋势一致;细胞周期和细胞凋亡检测结果显示,和normal组相比,OA组S期、G2/M期缩短,细胞比例明显下降,细胞早期和晚期凋亡比例增加,LV-Bmal1组S期、G2/M期延长,细胞比例增加,早期和晚期凋亡比例下降。结论 炎性软骨细胞中生物钟基因Bmal1可能参与调节细胞周期相关基因的表达。

基于网络药理学和实验验证探讨芝麻林素抗肝细胞癌作用及其机制

为了探究芝麻林素对肝细胞癌的抗肿瘤作用及其分子机制,通过网络药理学分析,预测芝麻林素与肝细胞癌的交集靶点,以及交集靶点富集的通路和生物学过程,然后检索SwissADME数据库分析芝麻林素的药代动力学。采用CCK8法检测细胞活性,采用Annexin V-FITC/PI双染法、流式细胞术、蛋白免疫印迹法、细胞迁移法分析芝麻林素处理后Huh-7细胞凋亡、周期阻滞及迁移抑制的作用与机制。结果表明,网络药理学分析可得交集靶点共64个,基因本体论功能共352个,京都基因与基因组百科全书信号通路共136个。体外细胞实验表明芝麻林素可以通过调控活性氧(reactive oxygen species,ROS)介导的丝裂原活化蛋白激酶/信号转导与转录激活因子3/核因子κB和磷脂酰肌醇3激酶(phosphatidylinositol 3-kinases,PI3K)/蛋白激酶B(protein kinase B,AKT)信号通路,诱导Huh-7细胞线粒体依赖性凋亡与G2/M期阻滞。同时,芝麻林素通过ROS介导的PI3K/AKT/糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)/β-连环蛋白信号通路抑制细胞迁移。最后,SwissADME分析结果表明芝麻林素具有良好的类药性。综上,芝麻林素可以通过ROS介导的信号通路诱导Huh-7细胞发生细胞凋亡、周期阻滞与抑制迁移作用。

达沙替尼基于PI3K/AKT信号通路调节乳腺癌细胞生物学行为

目的:基于PI3K/AKT信号通路探讨达沙替尼(dasatinib,DAS)对乳腺癌MCF-7细胞生物学行为的影响。方法:分别使用噻唑蓝(methyl thiazolyl tetrazolium,MTT)法、Transwell法、流式细胞术和Western blotting法检测DAS不同浓度(0、2、6、10μmol/L)作用下MCF-7细胞增殖、侵袭和迁移、细胞凋亡以及PI3K/AKT信号通路相关蛋白表达情况。同时设置对照组(溶媒对照)、DAS组(DAS 10μmol/L)、PI3K抑制剂组(LY29400220μmol/L)、联合组(DAS 10μmol/L+LY29400220μmol/L),比较各组细胞增殖、侵袭和迁移、细胞凋亡以及PI3K/AKT信号通路相关蛋白表达情况。结果:随着DAS作用浓度的升高,MCF-7细胞增殖抑制率和细胞凋亡率升高(P<0.05),侵袭细胞数、迁移细胞数和PI3K、p-PI3K、p-AKT蛋白表达降低(P<0.05)。与对照组相比,DAS组、PI3K抑制剂组、联合组MCF-7细胞增殖抑制率和细胞凋亡率升高(P<0.05),PI3K、p-PI3K、p-AKT蛋白表达降低。与PI3K抑制剂组、DAS组相比,联合组MCF-7细胞增殖抑制率和细胞凋亡率升高,PI3K、p-PI3K、p-AKT蛋白表达降低(P<0.05)。结论:DAS能抑制乳腺癌MCF-7细胞增殖、侵袭和迁移能力,诱导细胞凋亡,其机制可能与调控PI3K/AKT信号通路有关。

桃叶珊瑚苷抑制人肝癌细胞系HepG2增殖

目的探讨桃叶珊瑚苷(AU)对人肝癌细胞系HepG2增殖、凋亡和细胞周期的影响及其作用机制。方法体外培养HepG2细胞,CCK-8法筛选AU的最佳给药浓度。随机将HepG2细胞分为对照组、AU 12.5 mg/L组(AU L组)、AU 62.5 mg/L组(AU H组)和AU H+Akt通路激动剂(SC79)组(AU H+SC79组),观察各组细胞增殖状态。5-乙炔基-2′脱氧尿嘧啶核苷(EDU)法检测细胞增殖;流式细胞测量术检测细胞凋亡和细胞周期;Western blot检测磷酸化-蛋白激酶B(p-Akt)、Akt、p-MDM2、MDM2、p-p53、p53蛋白表达水平。结果选择浓度为12.5、62.5 mg/L的AU进行后续实验。与0 mg/L AU比较,AU L组、AU H组细胞增殖显著降低(P<0.05);与对照组比较,AU L、AU H组悬浮和脱落细胞逐渐增多,细胞皱缩变圆,G0/G1期细胞占比、EDU阳性染色细胞比例以及p-Akt/Akt、p-MDM2/MDM2蛋白表达水平下降,S和G2/M期细胞占比、细胞凋亡率以及p-p53/p53蛋白表达水平升高(P<0.05);与AU H组比较,AU H+SC79组上述变化得到改善(P<0.05);移植瘤裸鼠接受AU治疗后,瘤体体积和质量均下降。结论AU可能通过调控Akt/MDM2/p53信号通路抑制人肝癌细胞增殖,诱导细胞周期阻滞与细胞凋亡。

芍药苷调节丝氨酸/苏氨酸激酶/鼠双微基因2/p53信号通路对弥漫大B细胞淋巴瘤细胞增殖、凋亡和细胞周期的影响实验研究

目的:探讨芍药苷(PAE)调节丝氨酸/苏氨酸激酶(AKT)/鼠双微基因2(MDM2)/p53信号通路对弥漫大B细胞淋巴瘤(DLBCL)细胞增殖、凋亡和细胞周期的影响。方法:以OCI-LY3细胞为研究对象,分别设置PAE低浓度组(15μmol/L PAE)、PAE中浓度组(30μmol/L PAE)、PAE高浓度组(60μmol/L PAE)、PAE高浓度+SC79(AKT激活剂)组(60μmol/L PAE+8μg/ml SC79),同时以未经处理的细胞为对照组。48 h后,分析细胞增殖、克隆形成能力及细胞周期和凋亡变化,检测AKT/MDM2/p53信号通路相关蛋白的表达水平。结果:与对照组比较,PAE低浓度组、PAE中浓度组、PAE高浓度组细胞凋亡率、p53表达、G_(0)/G_(1)期增加,细胞克隆形成数和细胞增殖率,p-AKT/AKT和p-MDM2/MDM2表达水平,以及G_(2)/M、S期降低(均P<0.05)。与PAE高浓度组比较,PAE高浓度+SC79组细胞凋亡率、p53表达、G_(0)/G_(1)期降低,细胞克隆形成数和细胞增殖率,p-AKT/AKT和p-MDM2/MDM2表达水平,以及G_(2)/M、S期增加(均P<0.05)。结论:PAE通过抑制AKT/MDM2上调p53表达,抑制DLBCL细胞增殖、细胞周期进展,诱导其凋亡。