Synergistic Anti-cancer Effect and Mechanism of FZKA Decoction Combined with Gefitinib on A549 Cells / 中国实验方剂学杂志

Objective::To observe the effect of Fuzheng Kangai (FZKA) decoction combined with gefitinib on the cells proliferation, apoptosis, invasion and metastasis of human lung adenocarcinoma A549 cells in vitro and in vivo, and relevant mechanisms. Method::The A549 cell proliferation of the control group, FZKA decoction groups (0.2, 0.4, 0.8, 1.6, 3.2 g·L-1), Gefitinib groups (10, 20, 40, 60, 80, 100 μmol·L-1) for 24, 48, 72 hours, and FZKA decoction (2 g·L-1) combined with Gefitinib (10 μmol·L-1) groups for 24 hours was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. The changes of cell apoptosis, invasion and metastasis abilities of A549 cells were analyzed by flow cytometry, Wound Healing, transwell invasion assay. Western blot assay was used to examine the protein expressions of cleaved Caspase-3, B-cell lymphoma-2 (Bcl-2), B-cell lymphoma-2 associated X (Bax), F-box and WD repeat domain-containing (FBW7) and myeloid cell leukemia-1 (MCL-1) in vitro. Result::Compared with control group, FZKA decoction group and Gefitinib group inhibited the cell proliferation, cell apoptosis, cell invasion and metastasis abilities in a dose-dependent and time-dependent manner, and improve the protein expressions of Bax, Caspase-3, FBW7, but decreased the protein expressions of Bcl-2, MCL-1 (P<0.05). Compared with treatment with Gefitinib alone, FZKA combined with Gefitinib inhibited the proliferation of A549 cells, and induced apoptosis more significantly (P<0.05). Compared with treatment with Gefitinib alone, the cell scratch healing and invasion abilities were significantly reduced after combined treatment (P<0.05). FZKA decoction combined with Gefitinib up-regulated Bax, Caspase-3 and FBW7 protein expressions, and down-regulated Bcl-2 and MCL-1 protein expressions compared with treatment with Gifitinib alone (P<0.05). Conclusion::FZKA decoction combined with Gefitinib can inhibit the proliferation, invasion and metastasis, and induce apoptosis on A549 cells. The mechanism may be associated with the FBW7/MCL-1 pathway.

Myeloid cell leukemia-1 protein expression and myeloid cell leukemia-1 gene amplification in non small cell lung cancer

Background: Myeloid cell leukemia-1 (Mcl-1) is a member of the B-cell lymphoma 2 family known to play a significant role in the regulation of apoptosis. Mcl-1 expression has been studied in nonsmall cell lung cancer (NSCLC) cell lines but has not been previously evaluated as a prognostic factor in clinical samples. Materials and Methods: Formalin-fixed, paraffin-embedded sections from 119 NSCLC, including 33 squamous cell carcinomas (SCC), 55 adenocarcinomas (AC), and 31 either pure adenocarcinoma in situ (AIS) or AC with lepidic features were immunostained by an automated method with rabbit polyclonal Mcl-1. Cytoplasmic Mcl-1 (cMcl-1) immunoreactivity was scored based on intensity and percentage of positive tumor cells in both tumor and adjacent benign epithelium in each case. MCL1 amplification was determined by hybrid capture-based comprehensive genomic profiling (CGP) on a separate cohort of 9393 NSCLC samples. Results: Intense diffuse cMcl-1 overexpression was noted in 35/119 (29%) tumors overall and correlated with tumor type (52% AIS vs. 31% AC vs. 6% SCC, P < 0.0001), tumor grade (48% grade 1 vs. 14% grade 2 vs. 31% grade 3, P = 0.007), small tumor size (36% ?3.0 cm vs. 16% >3.0 cm, P = 0.016), and lengthened survival within the AIS subgroup (100% alive vs. 42% expired, P = 0.018) while showing a trend toward correlation with nonrecurrent disease overall (32% nonrecurrent vs. 11% recurrent, P = 0.072) and within the AC subgroup (33% nonrecurrent vs. 0% recurrent, P = 0.092). MCL1 amplification was identified in 569 (6%) of 9393 NSCLC by CGP. Conclusions: cMcl-1 overexpression appears to occur independently from MCL1 gene amplification in NSCLC and correlates with AIS histologic type, lower tumor grade, smaller tumor size, nonrecurrent disease, and increased survival.

To study the correlation of peripheral blood F-box and WD repeat domain-containing domain protein7 and myeloid cell leukemia-1 expression levels with the prognosis of Icotinib administration in elderly patients with advanced non-small-cell lung cancer / 中华老年医学杂志

Objective To study the correlation of the prognosis of Icotinib administration with the expression levels of F-box and WD repeat domain-containing 7(FBW7) and myeloid cell leukemia-1 (MCL-1) in peripheral blood in elderly patients with advanced non-small-cell lung cancer.Methods A total of 76 patients aged 60 years or over diagnosed with non-small-cell lung cancer(NSCLC) with EGFR-sensitive mutations and under Icotinib treatment were enrolled in this study.FBW7 and MCL-1 mRNA expression levels in peripheral blood were detected by real-time quantitative PCR(RT-QPCR).The correlation of FBW7 and MCL-1 expression levels with clinical and histological parameters,overall survival (OS),and progression-free-survival (PFS) was analyzed.Results The FBW7 expression level and the MCL-1 expression level were negative correlated(r =-0.37,P ＜0.001).High FBW7 expression levels and low MCL-1 expression levels in peripheral blood were associated with improved therapeutic efficacy of Icotinib (P＜0.001) and extended OS and PFS.Cox regression analysis showed that the expression levels of FBW7 and MCL-1 in peripheral blood were independent influencing factors for OS and PFS.Conclusions Patients with high FBW7 expression levels and low MCl-1 expression levels are more likely to benefit from Icotinib treatment.Expression levels for either factor can be used as a predictive indicator for the effectiveness of Icotinib and provide guidance for its clinical use.

Expression and Significance of Mcl-1 Gene in Hepatocellular Carcinoma / 现代检验医学杂志

Objective To investigate the expression and significance of myeloid cell leukemia-1 (Mcl-1 )gene and protein in Hepatocellular Carcinoma(HCC).Methods The expression of Mcl-1 was detected respectively by Reverse Transcription-Polymerase Chain Reaction (RT-PCR),Western blot and ENVISION immunohistochemistry in 20 HCC specimens,19 liver cirrhosis(LC)specimens,and 12 control ones.Their relationship with clinical and pathological characteristics of HCC was investigated.Results The expression of Mcl-1 mRNA in the control group,LC group and HCC group was 0.52±0.17, 3.46±1.7,3.65±2.93,respectively.The level in HCC and LC group was statistically different compared with the control group,respectively (t=7.925,5.334,P<0.05).The relative expression of Mcl-1 protein in LC group (0.51±0.35)and HCC group (0.75±0.36)were significantly higher than that in the control group (0.21±0.19)(t=5.526,6.355,P<0.05).The positive expression rate of Mcl-1 in HCC group was 55.00% (11/20),significantly higher than that in the con-trol group 33.33% (4/12)(t=7.835,P<0.05).The positive expression of Mcl-1 was related to tumor necrosis and TNM staging (χ2=4.201,P<0.05).Conclusion Mcl-1 gene and protein are differentially expressed in HCC,LC and the control, which may be involved in the occurrence,development and malignant transformation of HCC.

Expression and clinical significance of MCL-1 and FBW7 proteins in breast cancer polyploid induced by spindle poisons / 天津医药

Objective To investigate the expression and clinical significance of myeloid cell leukemia-1 (MCL-1) and F-box and WD repeat domain-containing 7 (FBW7) in breast cancer polyploid induced by spindle poisons. Methods (1) Nocodazole spindle poison was used to treat breast cancer cell MDA-MB-231. The morphological changes of cells were ob?served under microscope, and cells were harvested in 0, 6, 12, 24, 48 and 72 h. The cell cycle and DNA-ploidy changes were examined by flow cytometry. The expressions of FBW7 and MCL-1 proteins were detected by Western blot assay. (2) A multikinase inhibitor (Sorafenib) with Nocodazole or Taxol was used to treat MDA-MB-231 cells. MCL-1 protein expression was detected by Western blot assay after 48 h treatment. The cell cycle and DNA-ploidy changes were examined by flow cy?tometry after 48 h treatment. MTT method was used to observe cell proliferation after 48 and 72 h treatment. Results (1)Af?ter treatment by Nocodazole, polyploid characteristics of large cell size and nucleus were appeared. The percentages of octa?ploid were (0.8±0.2)%, (8.5±2.3)%, (7.8±2.0)%, (9.9±0.9)%, (28.2±0.8)%and (35.1±4.9)%after 0, 6, 12, 24, 48 and 72 h treatment, showing the increasing trend in turn (P<0.001). The number of polyploidy (tetraploid and octaploid) cells was as high as (97.6±0.7)%after 48 h treatment. The expression level of FBW7 protein was decreased significantly but the expres?sion of MCL-1 protein was increased significantly after 48 h treatment. (2) After 48 h treatment, the expression level of MCL-1 protein, polyploidy percentage and cell proliferation decreased significantly in Nocodazole+Sorafenib group and Taxol+Sorafenib group compared with those of Nocodazole group and Taxol group (P<0.05). Conclusion The lower expression of FBW7 protein and over-expression of MCL-1 protein are correlated with the formation of breast cancer polyploidy. Sorafenib can reduce polyploid tumor cells by inhibiting MCL-1 protein expression.

Construction and identification of Mcl-1 gene shRNA expression plasmid in targeted silenced macrophages / 西安交通大学学报(医学版)

Objective To study the expressions of myeloid cell leukemia-1 (Mcl-1 ) gene in mouse macrophages Raw264.7 and human macrophages THP-1,to screen out the cell lines with high levels of expression as the experimental cells,and based on the screening results to construct the short hairpin RNA(shRNA)eukaryotic expression plasmid targeting mice Mcl-1 gene for transfection and further screen out the shRNA expression plasmid with the most obvious effect in silencing Mcl-1 gene.Methods Semi-quantitative PCR method was used to detect the expression of Mcl-1 mRNA in the two kinds of macrophages.Western blotting was adopted to detect the expressions of Mcl-1 proteins in the two kinds of macrophages.Three different gene loci for Mcl-1 shRNA fragments were designed with small molecules interfering RNA (siRNA)software.Eukaryotic expression plasmid Mcl-1 shRNA 1-3 carrying the shRNA fragments was constructed by a company.And the eukaryotic expression plasmid vector was transfected into scavenger cells Raw264.7 mice via through the liposome.The transfection results 24 h and 48 h after transfection were observed under the inverted fluorescence microscope;Mcl-1 mRNA and protein expression were detected by real time quantitative PCR and Western blot,respectively.Results The relative expression levels of Mcl-1 mRNA and protein in mouse macrophages Raw264.7 were significantly higher than those of human macrophages (P <0.05).The shRNA expression vector constructed within 24 h and 48 h could decrease Mcl-1 mRNA and protein levels in Raw264.7 cells,especially with the most obvious silencing effect at 48 h.The 48-h transfection group differed significantly from normal group,liposome group and negative control group (P <0.05).Compared with Mcl-1shRNA1and Mcl-1shRNA2,Mcl-1shRNA3 had the strongest effect in silencing Mcl-1mRNA and protein.Conclusion We have successfully screened out experimental Raw264.7 cells and Mcl-1 shRNA eukaryotic expression plasmid which has an obvious silencing effect targeting on Mcl-1 in mice macrophages Raw264.7.

Clinical significance of miR-26a-5p-regulated MCL-1 expression in pre-eclampsia / 中国病理生理杂志

AIM:To investigate the clinical significance of microRNA-26a-5p (miR-26a-5p)-regulated mye-loid cell leukemia-1 (MCL-1) expression in the development of maternal preeclampsia.METHODS:Plasma and placen-tal tissues were collected from 21 cases of normal pregnancy, 13 cases of maternal gestational hypertension, 15 cases of mild preeclampsia and 26 cases of severe preeclampsia.The levels of plasma and placental miR-26a-5p and placental MCL-1 mRNA were detected by real-time PCR.Western blotting analysis was used to determine the protein expression of placen-tal MCL-1.The clinical significance of the above parameters was also analyzed.RESULTS:miR-26a-5p expression gradu-ally increased(P<0.01) in the 4 groups of maternal plasma and placentas with the disease development, and the mRNA expression of MCL-1 was significantly reduced in the placentas (P<0.01), both showing a significant negative correlation (P<0.01).Meanwhile, the expression of miR-26a-5p and MCL-1 protein in the placental tissues was negatively correla-ted (P<0.01).The miR-26a-5p up-regulation in maternal plasma and placental tissues was negatively correlated with ges-tational age, maternal plasma albumin levels and fetal weight, while it was positively correlated with maternal blood pres-sure and urinary protein level (P<0.01), which was in contrary to the down-regulation of placental MCL-1.CONCLU-SION:Up-regulation of miR-26a-5p is involved in the occurrence and development of preeclampsia by down-regulation of MCL-1.

Neuroprotective effect of phosphatase and tensin homolog allele activity deleted in chromosome 10 inhibition on neuronal apoptosis after hypoxia-ischemia / 中华实用儿科临床杂志

Objective To investigate the mechanisms of neuroprotective roles of phosphatase and tensin homolog allele activity deleted in chromosome 10 (PTEN) inhibition on neuronal apoptosis after hypoxia-ischemia (HI)damage.Methods The cerebral cortical neurons of newbom Sprague-Dawley rats were cultured in vitro.Oxygen and glucose deprivation (OGD) model was established to imitate HI environment in vivo.Neurons were divided randomly into 3 groups:control group:neurons were treated with normal medium ; OGD group:neurons were treated with OGD for 3 h followed by reperfusion at 0.5,3.0,6.0,12.0,24.0,48.0 h ; PTEN inhibition group:before OGD treatment,neurons were pretreated with PTEN inhibitor,and then the neurons were collected at 24 h after reperfusion.Terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling staining was used to detect the apoptotic cells.Western blot was used to detect the expression of PTEN,p-PTEN,protein kinase B(Akt),p-Akt,synthesis of glucose kinase-3 betal (GSK-3β),p-GSK-3β and myeloid cell ceukemia-1 (Mcl-1).Results 1.As compared with control group,TUNEL positive cells increased after OGD observed by TUNEL staining (P ＜ 0.05).The expression of PTEN was increased,the expressions of p-PTEN,p-Akt,p-GSK-3 β,and Mcl-1 were significantly decreased after OGD (all P ＜ 0.05).However,Akt and GSK-3β remained unchanged at different time points after OGD(all P ＞ 0.05).2.As compared with OGD group,TUNEL positive cells were obviously reduced at 24 h after OGD in PTEN group (P ＜ 0.05).Although total PTEN,Akt,and GSK-3 β were not obviously changed in PTEN group(all P ＞ 0.05),the expressions of p-PTEN,p-Akt,p-GSK-3β,and Mcl-1 were significantly increased after OGD (all P ＜ 0.05).Conclusions HI can induce neuronal apoptosis,and the mechanisms of apoptosis may involve PTEN/Akt/GSK-3β/Mcl-1 pathway.The phosphorylation of Akt and GSK-3β can be increased via PTEN activity inhibition,while the ubiquitination of Mcl-1 would be decreased,and thus reduce the neuronal apoptosis.

Expression of Mcl-1 protein in human cervical lesions and its significance / 肿瘤

Objective. Myeloid cell leukemia-1 (Mcl-1) protein is one of the numbers in bel-2 superfamily, which plays an important role in controlling apoptosis, proliferation, and differentiation of tumor cells. This study was to detect the expression of Mcl-1 protein in different stages of human cervical lesions and to explore further their possible roles in the formation and progression of cervical cancer. Methods: The expression of Mcl-1 protein was detected in 83 paraffin-embedded specimens of the cervical tissues with immunohistochemical SP methods. Its clinical significance was analyzed associating with clinicopathological classification. Results: The positive rate of Mcl-1 protein expression was 4.0% 33.3%, and 74.2% in cervicitis, cervical intraepithelial neoplasia (CIN) and cervical cancer tissue, respectively. The difference was significant among the three groups (P 0.05). Conclusion: Over-expression of Mcl-I may play an important role in the initiation and development of cervical cancer.

Benzyl isothiocyanate induces apoptosis of human leukemia cells:molecular mechanism / 第三军医大学学报

Objective To explore the apoptotic effect of benzyl isothiocyanate(BITC) on human leukemia cells and investigate its related molecular mechanism.Methods Cells(U937,Jurkat and HL60) were exposed to BITC at various concentrations(0,2,4,6 or 8 ?mol/L) for 6 h or 12 h,or at 8 ?mol/L for different time intervals.The apoptosis was measured using flow cytometry.The apoptotic-related proteins,such as Caspase-3,poly ADP-ribose polymerase(PARP),myeloid cell leukemia-1(Mcl-1) and so on,were determined using Western blot assay.Results BITC induced apoptosis in human leukemia cells in dose-and time-dependent manners.BITC at a dose over 4 ?mol/L began to induce an increased expression of Caspase-3 protein and decreased expression of PARP in U937 cells,and when the dose was 8 ?mol/L,the changes reached their summits respectively.The expression of anti-apoptotic protein Mcl-1 was decreased in U937 cell after exposure of BITC.Conclusion BITC induces apoptosis in human leukemia cells including U937,Jurkat and HL60,and downregulation of Mcl-1 may play an important role in BITC-induced apoptosis.