scholarly journals Mycophenolate mofetil reduces epidural fibrosis by regulating TGF-β1/Smad2/3 axis to repress the proliferation, migration and differentiation of fibroblasts

2020 ◽  
Author(s):  
Dongming Zhu ◽  
Jie Zhang ◽  
Xiaobo Zhang ◽  
Yun Liu ◽  
Lianqi Yan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mycophenolate Mofetil ◽  
Western Blot ◽  
Cell Counting ◽  
Epidural Fibrosis ◽  
Transwell Assay ◽  
Scratch Assay ◽  
Inhibition Effect ◽  
Tgf Β1 ◽  
Wound Scratch Assay

Abstract Background Excessive fibroblasts proliferation is believed as a major reason in the process of epidural fibrosis, which is known as a troublesome complication of lumbar laminectomy clinically. Mycophenolate mofetil (MMF) is a kind of immunosuppressant, previous studies had shown that it has the function of preventing fibrosis, but the role and mechanism are still unclear. In this study, we determined the repressed effect of MMF on fibroblasts proliferation, migration and differentiation in surgical-induced epidural fibrosis and the underlying mechanism. Methods In vitro, the impacts of MMF on cell viability were measured by cell counting kit-8 (CCK-8) assay and the fibroblasts proliferation rates were determined by using EdU incorporation, cell cycle assays and western blot. Additionally, the impacts of MMF on fibroblasts migration and differentiation were analyzed by cell wound scratch assay, transwell assay, immunofluorescence analysis and western blot. In vivo, we built epidural fibrosis models in rats and locally applied MMF. Histological and immunohistochemical staining were used to determine the effect of MMF on reducing epidural fibrosis and fibroblasts functions. Results CCK-8 assay demonstrated that MMF repressed fibroblasts proliferation in a time- and a dose-dependent manner. EdU incorporation assay and cell cycle analysis proved the inhibition effect of MMF on the proliferation of fibroblasts. Cell wound scratch assay, transwell assay and immunofluorescence proved the inhibition effect of MMF on the migration and differentiation of fibroblasts. Western blotting analysis proved that MMF inhibited the expression of TGF-β1, p-Smad2 and p-Smad3. The expression of proliferation proteins, migration proteins and differentiation proteins were downregulated. In addition, histological and immunohistochemical stain showed that MMF reduces epidural fibrosis by repressing the proliferation, migration and differentiation of fibroblasts. Conclusion MMF could inhibit the proliferation, migration and differentiation of fibroblasts, it reduced surgical-induced epidural fibrosis as well. TGF-β1/Smad2/3 axis may be the latent mechanism in MMF reduced epidural fibrosis. It might provide a new notion for mitigating surgery-induced epidural fibrosis.

scholarly journals Chidamide, a Novel Histone Deacetylase Inhibitor, Displays Potent Antitumor Activity Against MDS Cells Mainly through JAK2/STAT3 Signaling Inhibition

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5233-5233 ◽  
Author(s):  
Chunkang Chang ◽  
Sida Zhao ◽  
Juan Guo ◽  
Youshan Zhao ◽  
Chengming Fei ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Histone Deacetylases ◽  
Conflicts Of Interest ◽  
Histone H3 ◽  
Cell Counting ◽  
Dependent Manner ◽  
Cytotoxic Effects ◽  
Hdac Activity ◽  
Stat3 Signaling

Abstract Background: Many studies have indicated that histone deacetylases (HDAC) activity is always increased in a lot of human tumors, and inhibition of HDAC activity are promising new strategies in the treatment of cancers. Chidamide (CS055/HBI-8000), a novel histone deacetylases inhibitor (HDACi) of the benzamide class, is currently under clinical trials. Despite emerging information on the effect of chidamide in multiple cancers, little is yet known about mechanism of action, and there were few reports about this drug's effects on myelodysplastic syndromes (MDS). In this study, we aimed to investigate the antitumor activities of chidamide on MDS cell line SKM-1 and to explore the possible mechanism. Methods: We treated MDS cells with different concentrations of chidamide. The effect of chidamide on HDAC activity of MDS cells was studied by using a HDAC Fluorometric Activity Assay Kit. The induction of histone acetylation was confirmed by detecting acetylated histone H3 and H4 using Western blot analysis. The effect of chidamide on the proliferation of MDS cells was analyzed by Cell Counting Kit (CCK-8) assay. Apoptosis were detected by Annexin V/propidium iodide (PI) double-labeled cytometry. Cell cycle was analyzed by a PI method. The acetylation levels of genes promoterassociated histone H3 and H4 were examined by chromatin immunoprecipitation (ChIP) and quantitative real-time PCR. Quantitativereal-time PCR and Western blot were used to detect the expression of signaling pathway factors (SOCS3, JAK2, STAT3,Bcl-XL,Bcl-2 and Mcl-1). Results: Our results demonstrated chidamide suppressed MDS cells growth in a time- and dose-dependent manner, and induced cell apoptosis and cell cycle arrest at G0/G1phase. Chidamide was able to significantly inhibit the HDAC activity and increase the acetylated histoneH3 and H4 of MDS cells. ChIP analysis further indicated that chidamide induced acetylated histones accumulation in the promoter of SOCS3.Moreover, chidamide upregulated the mRNA and protein expression ofSOCS3, and also significantly downregulated JAK2/STAT3 signaling. Further, we identified reduced expression of STAT3 downstream targets with treatment of chidamide, including Bcl-XL, Bcl-2 and Mcl-1, which may explain the cytotoxic effects of chidamide on MDS cells. Conclusion: These results demonstrate that chidamide possesses significant cytotoxic effects on MDS cells mainly through histone modifications of SOCS3 and consequently JAK2/STAT3 signaling inhibition. Therefore, Our data provide rationale for clinical investigation of chidamide in MDS. Disclosures No relevant conflicts of interest to declare.

scholarly journals Anticancer Activity of Modified Tongyou Decoction on Eca109 Esophageal Cancer Cell Invasion and Metastasis through Regulation of the Epithelial-Mesenchymal Transition Mediated by the HIF-1α-Snail Axis

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Yongsen Jia ◽  
Xin Yan ◽  
Ying Cao ◽  
Wei Song ◽  
Guangji Zhang ◽  
...  
Keyword(s):  
Esophageal Cancer ◽  
Western Blot ◽  
Cell Invasion ◽  
Epithelial Mesenchymal Transition ◽  
Invasion And Metastasis ◽  
Control Groups ◽  
Transwell Assay ◽  
Scratch Assay ◽  
Mesenchymal Transition ◽  
Related Proteins

Background. To explore the activity of Modified Tongyou Decoction (MTD) against Eca109 esophageal cancer (EC) cell invasion and metastasis and to ascertain the mechanism of its anticancer activity during the epithelial-mesenchymal transition (EMT) as mediated by the HIF-1α-Snail axis. Methods. Herbal compounds were prepared by ethanol extraction, and 6 herbs composing into MTD were dipped in water-free ethanol and filtered. The filtrate was collected and centrifuged. The remains were concentrated into a paste which was adjusted to 5000mg/mL concentration with DMSO. PBS was used to dilute the herbal solution to the half maximal inhibitory concentration. A hypoxic microenvironment was induced with CoCl2 in RPMI 1640 medium, in which Eca109 cells were cultured. The cytotoxicity of MTD was determined with CCK-8 assay. The activity of MTD against cell invasion and metastasis was explored with scratch assay and transwell assay. Western blot analysis was conducted to analyze the anticancer effects of MTD on the expression of HIF-1α-Snail axis- and EMT-related proteins. Quantitative RT-PCR was used to assess the mRNA expression of Snail. Immunofluorescence labeling was performed to examine how MTD affected the coexpression of Snail and HIF-1α. Results. The fifty percent inhibitory dose of MTD was 1410 μg/mL in the normoxic environment and 1823 μg/mL in the hypoxic environment based on the CCK-8 assay. The scratch assay showed that MTD significantly inhibited cell migration in both the normoxic and hypoxic microenvironments compared with the control groups ( P  < 0.05). The transwell assay showed that MTD significantly inhibited cell invasion in both the normoxic and hypoxic environments compared with the control groups ( P  < 0.05). Western blot showed that MTD significantly inhibited the expression of the HIF-1α, Snail, Vimentin, MMP-2, MMP-9, and VE-cadherin proteins and significantly induced the expression of E-cadherin in both the normoxic and hypoxic microenvironments compared with the control groups ( P  < 0.05). qRT-PCR indicated that MTD significantly inhibited Snail mRNA expression compared with that in the control groups ( P  < 0.05). Immunofluorescence assay showed that MTD significantly inhibited the coexpression of HIF-1α and Snail in both the normoxic and hypoxic microenvironments compared with the control groups ( P  < 0.05). Conclusion. MTD downregulated HIF-1α-Snail axis- and EMT-related proteins to inhibit EC cell invasion and metastasis in both the normoxic and hypoxic environments.

scholarly journals MO014DANHONG INJECTION INHIBITS LIPOPOLYSACCHARIDE-ENHANCED CELL PROLIFERATION OF RAT RENAL MESANGIAL CELLS VIA NF-ΚB SIGNALING PATHWAY

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Liu Hua ◽  
Lei Chen ◽  
Limin Wei ◽  
Hongli Jiang
Keyword(s):  
Cell Proliferation ◽  
Protein Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Inflammatory Mediators ◽  
Mesangial Cells ◽  
Inhibitory Effect ◽  
Cell Counting ◽  
Pyrrolidine Dithiocarbamate ◽  
Tgf Β1

Abstract Background and Aims Mesangioproliferative glomerulonephritis (MsPGN) is well known as one of the leading causes of end-stage renal failure (ESRD), especially in the Asian population. The principal characteristic of MsPGN is the over proliferation of glomerular mesangial cells (MCs) accompanied by expansion of the extracellular matrix (ECM). Our previous studies demonstrated that Danhong injection (DHI), is a traditional Chinese medicine injection, could improve the renal function and inhibit the MCs proliferation and ECM expansion in rats with MsPGN. However, the molecular mechanisms have not been fully elucidated. To explore the potential mechanisms of DHI in the treatment of MsPGN, we investigated the effects of DHI on lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation and its downstream inflammatory mediators, such as intercellular adhesion molecule-1 (ICAM-1), transforming growth factor-beta 1 (TGF-β1), inducible nitric oxide synthase (iNOS) and fibronectin (FN) protein expression in rat MCs. Method The rat MCs treated with different concentrations of DHI (0, 50, 100, 200, 500, 1000, and 2000 uL/L) for 12 h, then incubated with or without 100 ng/ml LPS for another 24 h. Subsequently, cell proliferation was determined by Cell Counting Kit-8 (CCK8). Furthermore, the rat MCs treated with low-dose DHI (250 uL/L), median-dose DHI (500 uL/L) and high-dose DHI (1000 uL/L) for 12 h or Ammonium pyrrolidine dithiocarbamate (PDTC) for 30 min before 24h treatment of LPS. Then the activation of NF-κB was detected by Western blot and immunofluorescence. The protein levels of ICAM-1, TGF-β1, iNOS and FN in rat MCs were detected by Western blot. Results The results of CCK-8 revealed that DHI significantly suppressed LPS-induced cell proliferation (shown in Fig.1). LPS stimulation resulted in a significant increment of p65 contents in nucleus and a decrement of p65 contents in cytoplasm in rat MCs compared with NC. PDTC and DHI exerted potent inhibitory effect on increasing expression of p65 in nucleus and decreasing in cytoplasm compared with LPS treatment group. The inhibitory effect on NF-κB nuclear translocation of DHI was in a dose- dependent manner (shown in Fig.2). The Western blot assay showed that the protein level of IκB-α in cytoplasm treated by LPS decreased significantly compared with that in control (shown in Fig. 3) and this decrement was significantly reversed by PDTC and DHI. In addition, the protein expression of ICAM-1, TGF-β1, iNOS and FN was also inhibited by PDTC and DHI (shown in Fig. 4). Conclusion DHI significantly repressed LPS-induced cell proliferation and FN expression in rat MCs through inhibiting the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators. The ameliorative effects of DHI on MsPGN might be associated with this inhibition effect on NF-B signaling pathway.

Knockdown of lysyl oxidase like 1 inhibits the proliferation and pro-fibrotic effects of Transforming growth factor-β1-induced hypertrophic scar fibroblasts

2021 ◽  
Author(s):  
Mengxia Ying ◽  
Yan Chen ◽  
Bo Yuan
Keyword(s):  
Cell Cycle ◽  
Western Blot ◽  
Wound Repair ◽  
Transforming Growth Factor ◽  
Lysyl Oxidase ◽  
Healing Process ◽  
Cell Cycle Distribution ◽  
Wound Healing Process ◽  
Matrix Deposition ◽  
Tgf Β1

Background: The excessive healing response during wound repair can result in hypertrophic scars (HS). Lysyl oxidase like 1 (LOXL1) has been reported to be associated with fibrosis via targeting TGF-β1 signaling. This study aimed to investigate the effect of LOXL1 on HS formation. Methods: The expression of LOXL1 in HS tissues and TGF-β1-induced HSFs was detected via RT-qPCR and western blot. LOXL1 was silenced in HSFs using transfection with short hairpin RNA (shRNA), then wound healing process including cell proliferation, cell cycle distribution, migration and extracellular matrix deposition along with Smad expression were measured by CCK-8, EdU staining, flow cytometry, transwell, immunofluorescence and western blot assays. Results: LOXL1 was up-regulated in HS tissues and TGF-β1-induced HSFs. Knockdown of LOXL1 inhibited proliferation and migration, but promoted cell cycle G0/G1 phase arrest in TGF-β1-induced HSFs. The increased expression of cyclin D1, CDK4, MMP2, MMP9, COL1A1, COL1A2, fibronectin, COL3A1, α-SMA, but decreased expression of p27, and the phosphorylation of Smad2 and Smad3 caused by TGF-β1 were also blocked by LOXL1 silence. Conclusions: Silence of LOXL1 could effectively inhibit TGF-β1-induced proliferation, migration and ECM deposition in HSFs via inactivating Smad pathway. Targeting LOXL1 may have future therapeutic implications for HS treatment.

scholarly journals Sevoflurane blocks glioma malignant development by upregulating circRELN through circRELN-mediated miR-1290/RORA axis

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xiaofang Kang ◽  
Hongxia Li ◽  
Zaiwang Zhang
Keyword(s):  
Cell Cycle ◽  
Tumor Growth ◽  
Glioma Cell ◽  
Orphan Receptor ◽  
Cell Counting ◽  
Matrix Metalloproteinase 2 ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Transwell Assay ◽  
Xenograft Models

Abstract Background Sevoflurane (Sev) has been reported to inhibit cancer development, and sevoflurane treatment in cancers is implicated with the deregulation of specific non-coding RNAs (ncRNAs). This study aimed to investigate the relationship between sevoflurane and circular RNA reelin (circRELN) in glioma. Methods The expression of circRELN, microRNA-1290 (miR-1290) and RAR-related orphan receptor A (RORA) was measured by quantitative real-time PCR (qPCR). Cell proliferative capacity was assessed by cell counting kit-8 (CCK-8) and colony formation assays. Cell apoptosis and cell cycle distribution were monitored by flow cytometry assay. Cell migration was assessed by wound healing assay and transwell assay, and cell invasion was assessed by transwell assay. The protein levels of matrix metalloproteinase-2 (MMP2), MMP9 and RORA were quantified by western blot. Tumor growth in vivo was assessed by Xenograft models. The binding relationship between miR-1290 and circRELN or RORA was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Results We found that circRELN expression was declined in glioma tissues and cells, while Sev treatment enhanced circRELN expression. In function, Sev notably inhibited glioma cell proliferation, migration and invasion and promoted apoptosis and cell cycle arrest, while circRELN knockdown reversed these effects. MiR-1290 served as a target of circRELN, and glioma cell malignant phenotypes recovered by circRELN knockdown were partly repressed by miR-1290 deficiency. In addition, RORA was a target of miR-1290, and glioma cell malignant phenotypes promoted by miR-1290 restoration were partly blocked by RORA overexpression. CircRELN regulated RORA expression by targeting miR-1290. In Xenograft models, Sev inhibited tumor growth by upregulating circRELN. Conclusion Sev blocked the progression of glioma by increasing circRELN expression, and circRELN played roles in glioma partly by regulating the miR-1290/RORA network.

scholarly journals Inhibition of autophagy enhanced chemosensitivity in cisplatin resistant hypopharyngeal squamous carcinoma cells

2020 ◽  
Author(s):  
pin dong ◽  
Jia Zhang ◽  
Wei Mao ◽  
Yuying Liu ◽  
Jian Ding ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Multidrug Resistance ◽  
Survival Rate ◽  
Western Blot ◽  
Cell Line ◽  
Cell Counting ◽  
G1 Arrest ◽  
Fadu Cell ◽  
Squamous Carcinoma Cells ◽  
Fadu Cells

Abstract Background: Hypopharyngeal carcinoma is characterized by high degree of malignancy. The most common pathological type is squamous cell carcinoma (HSCC). It has been confirmed that high autophagy level promotes the development of hypopharyngeal cancer in recent years. Clinical researches have reported that high autophagy level often caused insensitivity to chemotherapy, a common phenomenon that greatly reduces therapeutic effect in cisplatin-resistant tumor cell lines. Therefore, exploring internal mechanisms of autophagy on cisplatin resistant HSCC is necessary for founding theoretical basis for synergistic antitumor drugs by interfering with autophagy.Methods: Part I: Cisplatin-resistant FaDu cell line was established and cultured. Cell counting kit-8 was used to detect drug resistance. Inverted microscope was used to observe the morphological changes at different concentrations, then the survival rate was calculated. After MDC staining, the autophagic vacuoles were observed by fluorescence microscopy. The expression of Beclin1 from each group was confirmed by RT-PCR and Western blot method. Part II: Beclin1 was knocked down by plasmid transfection, autophagy inhibitor 3-MA was applied for cisplatin-resistant cells intervention. Cell cycle was detected using flow cytometry assay, apoptosis with necrosis was detected by staining with propidium iodide (PI). CCK-8 was used to observe the cell survival rate in each group. The expression of autophagy-related gene Beclin1,LC3I,LC3II,Atg-5 and P62 in each group was verified by Western blot analysis.Results: Cisplatin-resistant FaDu cell line can be stably constructed by cisplatin intervention. Compared with normal group, autophagy and its related protein Beclin1 expression was enhanced in cisplatin resistant FaDu cells. Autophagy inhibition group showed significant cell cycle changes, mainly manifested by G1 arrest, increased apoptosis rate and significantly decreased survival rate at 24h level. Furthermore, Western blot showed that expression of Beclin1, lc3i, lc3ii, atg-5 protein decreased significantly after the inhibitor used, while the expression of p62 up-regulated, which also confirmed autophagy flow was blocked.Conclusion: Our work confirmed high autophagy level is important for the cisplatin-resistance of HSCC and insensitivity to chemotherapy. The use of 3-MA and Beclin 1 inhibition can significantly reduce autophagy level of cisplatin-resistant FaDu cells, arresting its cell cycle, promote apoptosis and reverse the multidrug resistance condition. These results provide the experimental basis for overcoming multidrug resistance through combination chemotherapy.#These authors contributed equally to this work.

scholarly journals Broussochalcone A Induces Apoptosis in Human Renal Cancer Cells via ROS Level Elevation and Activation of FOXO3 Signaling Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-17
Author(s):  
Han Ki Lee ◽  
Hyo Sun Cha ◽  
Myeong Jin Nam ◽  
Kyungmoon Park ◽  
Yung-Hun Yang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Western Blot ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Renal Cancer ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Counting ◽  
Apoptotic Effects

Broussochalcone A (BCA) is a chalcone compound extracted from the cortex of Broussonetiapapyrifera (L.) Ventenat that exerts various effects, such as potent antioxidant, antiplatelet, and anticancer effects. However, the effects of BCA against cancers have been seldom studied. This study is aimed at demonstrating the apoptotic mechanisms of BCA in A498 and ACHN cells, which are two types of human renal cancer cell lines. MTT, cell counting, and colony formation assays indicated that BCA treatment inhibited cell viability and cell growth. Further, cell cycle analysis revealed that BCA induced cell cycle arrest at the G2/M phase. Annexin V/PI staining and TUNEL assays were performed to determine the apoptotic effects and DNA fragmentation after treatment with BCA. Based on western blot analysis, BCA induced the upregulation of cleaved PARP, FOXO3, Bax, p21, p27, p53, phosphorylated p53 (ser15, ser20, and ser46), and active forms of caspase-3, caspase-7, and caspase-9 proteins, but downregulated the proforms of the proteins. The expression levels of pAkt, Bcl-2, and Bcl-xL were also found to be downregulated. Western blot analysis of nuclear fractionation results revealed that BCA induced the nuclear translocation of FOXO3, which might be induced by DNA damage owing to the accumulation of reactive oxygen species (ROS). Elevated intracellular ROS levels were also found following BCA treatment. Furthermore, DNA damage was detected after BCA treatment using a comet assay. The purpose of this study was to elucidate the apoptotic effects of BCA against renal cancer A498 and ACHN cells. Collectively, our study findings revealed that the apoptotic effects of BCA against human renal cancer cells occur via the elevation of ROS level and activation of the FOXO3 signaling pathway.

scholarly journals Knockdown of DGKZ induces apoptosis and G2/M phase arrest in human acute myeloid leukemia HL-60 cells

2018 ◽  
Author(s):  
Changhu Dong ◽  
Hong Li ◽  
Yanning Tian ◽  
Xiang Li ◽  
Bing Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Myeloid Leukemia ◽  
Western Blot ◽  
Myeloid Leukemia ◽  
Cell Counting ◽  
Cycle Phase ◽  
Cycle Arrest ◽  
M Phase ◽  
Human Acute Myeloid Leukemia ◽  
Acute Myeloid

AbstractDiacylglycerol kinase zeta (DGKZ) is associated with the pathogenesis of a variety of malignant diseases, but its biological function on acute myeloid leukemia (AML) has not been explored. This study was aimed to analyze apoptosis induced by Knockdown of DGKZ and its mechanism in human acute myeloid leukemia HL-60 cells.In the present study qRT-PCR was used to detect the expression of DGKZ in HL-60, THP-1, K562, H9, Jurkat and CD34 cell lines. DGKZ-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. Cell Counting Kit-8 (CCK-8) assay was used to determine the viability of HL-60 cells DGKZ knocked down. Apoptosis and cell cycle phase of HL-60 cells after DGKZ knockdown were evaluated by flow cytometry. Western blot analysis was performed to investigated the protein expression related to apoptosis and cell cycle. Results showed DGKZ expression were stable and higher in Jurkat, HL-60, THP-1,K562 leukemia cells than those of H9 and CD34 cells. Compared with cells of the shCtrl group, DGKZ was markedly knocked down in cells which were transfected with lentivirus encoding shRNA of DGKZ in HL-60 cells. DGKZ knockdown significantly inhibited the proliferation and induced cycle arrest at the G2/M phase in HL-60 cells. Western blot results indicated the expressions of caspase-3, caspase-8, and survivin markedly increased in HL-60 cells after knockdown of DGKZ. The results suggest Knockdown of DGKZ can inhibit proliferation of acute myeloid leukemia HL-60 cell caused cell cycle arrest at the G2/M phase through caspases pathway.

scholarly journals Berbamine Inhibits Cell Proliferation, Migration, and Invasion and Induces Cell Apoptosis of A549 Cells Via Regulating C-Maf, PI3K/Akt and MDM2-P53 Pathways

2020 ◽  
Author(s):  
Lili Liu ◽  
Zhiying Xu ◽  
Binbin Yu ◽  
Li Tao ◽  
Ying Cao
Keyword(s):  
Cancer Cells ◽  
A549 Cells ◽  
Migration And Invasion ◽  
Dependent Manner ◽  
Transwell Assay ◽  
Scratch Assay ◽  
Western Blotting Assay ◽  
Wound Scratch Assay

Abstract Background To investigate the influences of berbamine (BBM) on the cell viability, proliferation, and migration of A549 cells in vitro and in vivo, and explore the possible mechanisms.MethodsAfter the A549 cells were treated with BBM, the cell viability and proliferation of the cancer cells were detected by MTT assay, EdU assay, and colony formation assay. Migration and invasion of cancer cells were illustrated through wound scratch assay and transwell assay. Apoptosis of cancer cells was evaluated by trypan blue dye exclusion assay and elisa assay. Beside, the expression of PI3K/Akt signal pathway-related proteins and c-Maf were detected employing western blotting assay. Xenografted model of NSCLC was used to detect the effect of BBM on tumor growth and metastasis in vivo.ResultsMTT assay showed that BBM inhibited the viability of A549 cells in a concentration-dependent manner and time-dependent manner. The results from the colony formation assay and EdU assay revealed that BBM (10 µM) could significantly inhibit the proliferation of A549 cells (P＜0.001). And BBM (10 µM) significantly inhibited the migration and invasion in the wound scratch assay and transwell assay (P＜0.05). Trypan blue assay and elisa assay indicated that BBM (20 µM) significantly induced apoptosis of A549 cells. The nude mice assay manifested the tumor volume was significantly shrank by BBM (20 mg/kg) (P＜0.05). Western blotting assay showed that the PI3K/Akt and MDM2-p53 signaling pathways were inhibited by BBM, and the expression of c-Maf was downregulated by BBM. ConclusionsBBM could inhibit the proliferation and metastasis, and induce apoptosis of A549 cells in vitro and in vivo, these effects may be achieved by reducing the expression of c-Maf and regulating the PI3K/Akt and MDM2-p53 pathways.

scholarly journals Down-regulation of CCNE1 expression suppresses cell proliferation and sensitizes gastric carcinoma cells to Cisplatin

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Chao Zhang ◽  
Qiang Zhu ◽  
Jianzhong Gu ◽  
Shan Chen ◽  
Qian Li ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Gastric Carcinoma ◽  
Western Blot ◽  
Cell Lines ◽  
Survival Curve ◽  
Lymphatic Invasion ◽  
Cell Counting ◽  
Down Regulation ◽  
Qrt Pcr

AbstractA novel oncogene CCNE1 (cyclin E) is considered to be associated with the development of various tumor types, its role in gastric carcinoma (GC) is little studied and the effect of CCNE1 on chemotherapy also remains unclear. We recruited 55 cases of GC tissues and corresponding normal tissues. Immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and Western blot analysis were performed to detect the expression of CCNE1. We also examined the expression of CCNE1 in gastric mucosal GES-1 cells and five GC cell lines. Silencing CCNE1 was used to assess its effect on proliferation and cell cycle in MGC-803 and NCI-N87 cells, as performed by Cell counting kit-8 (CCK-8) and flow cytometry assay. Meanwhile, cell cycle related genes were also detected through qRT-PCR and Western blot. The results showed CCNE1 up-regulation mainly expressed in GC tissues and GC cell lines, also was associated with tumor node metastasis (TNM) stage and lymphatic invasion. Three-year survival curve analysis showed CCNE1 with high expression had a poor prognosis. Silencing CCNE1 significantly reduced cell viability in 48 h, cultured and arrested cell cycle in G1 phase, moreover, Cyclin A, D1 and C-myc all revealed down-regulation in both MGC-803 and NCI-N87 cells. CCNE1 expression was significantly increased at low and moderate concentrations of Cisplatin. Down-regulation of CCNE1 expression would remarkably promote cell apoptosis induced by Cisplatin, and regulate the rate of Bax/Bcl-2. Down-regulation of CCNE1 expression could inhibit cell proliferation and enhance GC cells sensibility to Cisplatin, possibly involving the regulation of Bcl-2 family.

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