Phosphorylation of Cofilin-1 Enhances Paclitaxel Resistance of Epithelial Ovarian Cancer Cells by Inhibiting Apoptosis / 生物医学与环境科学（英文）

Objective@#To investigate the molecular mechanism of high phosphorylation levels of cofilin-1 (p-CFL-1) associated with paclitaxel resistance in epithelial ovarian cancer (EOC) cells.@*Methods@#Cells displaying varying levels of p-CFL-1 and CFL-1 were created by plasmid transfection and shRNA interference. Cell inhibition rate indicating paclitaxel efficacy was assessed by Cell Counting Kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry and protein levels were detected by western blotting. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of phosphokinases and phosphatases of CFL-1. Survival analysis evaluated the correlation between the prognosis of EOC patients and the levels of p-CFL-1 and slingshot-1 (SSH-1).@*Results@#High levels of p-CFL-1 were observed in EOC cells that survived treatment with high doses of paclitaxel. SKOV3 cell mutants with upregulated p-CFL-1 showed impaired paclitaxel efficacy, as well as decreased apoptosis rates and pro-survival patterns of apoptosis-specific protein expression. Cytoplasmic accumulation of p-CFL-1 inhibited paclitaxel-induced mitochondrial apoptosis. SSH-1 silencing mediated CFL-1 phosphorylation in paclitaxel-resistant SKOV3 cells. Clinically, the high level of p-CFL-1 and the low level of SSH-1 in EOC tissues were closely related to chemotherapy resistance and poor prognosis in EOC patients.@*Conclusion@#The SSH-1/p-CFL-1 signaling pathway mediates paclitaxel resistance by apoptosis inhibition in EOC and is expected to be a potential prognostic predictor.

Detection of sputum cofilin-1 as indicator of malignancy

Cofilin-1 (CFL1), a small protein of 18 kDa, has been studied as a biomarker due to its involvement in tumor cell migration and invasion. Our aim was to evaluate CFL1 as an indicator of malignancy and aggressiveness in sputum samples. CFL1 was analyzed by ELISA immunoassay in the sputum of 73 lung cancer patients, 13 cancer-free patients, and 6 healthy volunteers. Statistical analyses included ANOVA, ROC curves, Spearman correlation, and logistic regression. Sputum CFL1 levels were increased in cancer patients compared to cancer-free patients and volunteers (P<0.05). High expression of sputum CFL1 was correlated to T4 stage (P=0.01) and N stage (P=0.03), tobacco history (P=0.01), and squamous cell carcinoma histologic type (P=0.04). The accuracy of sputum CFL1 in discriminating cancer patients from cancer-free patients and healthy volunteers were 0.78 and 0.69, respectively. CFL1 at a cut-off value of 415.25 pg/mL showed sensitivity/specificity of 0.80/0.70 in differentiating between healthy volunteers and cancer patients. Sputum CFL1 was also able to identify cancer-free patients from patients with lung cancer. The AUC was 0.70 and, at a cut-off point ≥662.63 pg/mL, we obtained 60% sensitivity and 54% specificity. Logistic regression analysis controlled for tobacco history, histologic types, and N stage showed that cancer cell-associated CFL1 was an independent predictor of death. Smoker patients with squamous cell carcinoma, lymph node metastasis and sputum CFL1>1.475 pg/mL showed augmented chance of death, suggesting lung cancer aggressiveness. CFL1 presented diagnostic value in detecting lung cancer and was associated to tumor aggressiveness.

The relationship between phosphorylation at Ser3 of cofilin1 and Taxol resistance of ovarian cancer in elderly female patients / 中华老年医学杂志

Objectives To analyze the relationship between phosphorylation of serine-3 of cofilin1 and Taxol resistance of ovarian cancer and to evaluate the prognostic value of cofilin1 phosphorylation in estimating ovarian cancer survival using clinical samples.Methods Wild type (WT) plasmids with over-expression of wild cofilin1,S3L plasmids with over-expression of cofilin1 and inhibited phosphorylation at serine-3,and siRNA-C plasmids with down-regulation of cofilin1 were constructed using molecular biology techniques.After the SKOV3 and SK-TR30 cell-lines were transfected with these plasmids,changes in biological characteristics and drug resistance after instant transfection were compared.Fifty-one elderly female patients with microscopically confirmed ovarian cancer,who had received chemotherapy with Taxol and cis-platinum (TC)after surgery,including 30 chemo-sensitive and 21 chemo-resistant cases,were recruited in this study.Immunohistochemical methods were used to detect the expression of cofilinl and phosphorylated cofilin in tissue sections from the two groups.Progression free survival(PFS)was analyzed.Results Compared with the control group,the proportion of cells increased at the G0/G1 phase(P=0.034)and decreased at the S phase (P=0.031),and the apoptosis rate decreased(P=0.020),in SKOV3 cells with high expression of cofilin1.However,these characteristics disappeared when the wild type was replaced with the inactive mutant S3L.When cofilin1 expression was down-regulated in SK-TR30 cells,the proportion of cells at the G0/G1 phase decreased(P=0.020).The expression of cofilin1 was detected in 56.7 ％ (17/30)and 57.1％ (12/21),respectively,sections of ovarian tumor tissues of the chemo-sensitive and chemoresistant groups,and there was no statistic difference (x2=0.332,P =0.943)between the two groups.The expression of phosphorylated cofilin was much higher in the chemo-resistant group (85.7％ or 18/21)than in the chemo-sensitive group (53.3％ or 16/30),and the difference was statistically significant(x2=5.829,P=0.016).The higher expression of phosphorylated cofilin was also correlated with shorter PFS(x2 =21.440,P＜0.01,95％ CI:0.068-0.883),especially in the chemo-sensitive group.Conclusions Serine-3 phosphorylation status of cofilin 1 is associated with paclitaxel resistance in ovarian cancer,but the underlying mechanisms of regulation need further investigation.

Effects of RNAi-mediated Cofilin-1 Gene Silencing on proliferation and invasiveness in hepatocellular carci-noma Huh-7 cells / 医学研究生学报

[Abstract ] Objective Cofilin-1 is involved in the pathogenesis of various tumours .However, the expression and effect of Cofilin-1 in hepatocellular carcinoma is not clear .The aim of this study is to observe the Cofilin-1 gene expression in human hepatocel-lular carcinoma (HCC) tissues, and to explore the effect of Cofilin-1 gene expression on invasion and metastasis of HCC HuH-7 cells. Methods Real-time quantitative PCR was used to assess the Cofilin-1 gene expression in human HCC tissues and normal tumor-ad-jacent tissues.The specific small interfering RNA ( siRNA) of Cofilin-1 sequence was synthetized in vitro , and was transfected into HCC HuH-7 cells using liposome transfection.The experiment was divided into Cofilin-1-siRNA group, Ctrl-siRNA group and un-transfected group.Western blot assay was used to detect the protein expression of Cofilin-1.Migration and invasion experiments in vitro were used to investigate the invasive ability of transfected cells. Results Compared with the adjacent liver tissue , Cofilin-1 gene ex-pression in human liver cancer tissue was significantly increased (0.698 ±0.156 vs 3.523 ±0.412, P and in untransfected group (0.961 ±0.020) (P<0.05).The results of migration and invasion experiments in vitro showed that the amount of migration and invasion cells in Cofilin-1-siRNA group were significantly lower than Ctrl-siRNA group or untransfected group (58.50 ±1.78 vs 79.00 ±1.33, 74.50 ±1.35,P<0.05; 36.50 ±0.83 vs 60.20 ±1.60, 51.50 ±1.14, P<0.05). Conclusion Cofilin-1 is highly expressed in HCC, and the invasion and metastasis of HCC HuH-7 cells is suppressed by inhibiting the Cofilin-1 gene expression.

Effect of baicalin on proliferation and migration activity in human skin SCC cells / 中国药理学通报

Aim To investigate the effect of baicalin on cell proliferation and cell migration in human skin SCC A431 cell line. Methods The A431 cells were incu- bated with 50 mg·L-1 baicalin. The protein level of cofilin-1 was assayed by Western blot. Cofilin-1 specific siRNA fragment was designed , synthesized and trans- fected into A431 cells. The proliferative activity and migration ability of cells were assessed by CCK8 assay and scratch wound healing assay separately. ResultsWestern blot results showed that baicalin treatment in-hibited the cofilin-1 protein expression to 49.3% com-pared with the control group. Single baicalin treatment and cofilin-1 silencing could drease the A431 cell growth and migration. And cofilin-1 silencing signifi- cantly enhanced the efficacy of baicalin. Conclusions Baicalin could significantly inhibit the tumor cell's growth and migration in the A431 cell line. And cofi-lin-1 might become the potential target gene to enhance the effect of anticancer drugs.

Diallyl disulfide inhibits migration and invasion in human colon cancer SW480 cells through Rac1-ADF/cofilin1 pathway / 中国肿瘤临床

Objective:This work aims to investigate diallyl disulfide (DADS) inhibition of cell migration and invasion in human colon cancer SW480 cells through the Rac1-ADF/cofilin1 pathway. Methods:The potential of cell migration and invasion was examined by scratch healing assay and transwell membrane assay. The expression of Rac1-ADF/cofilin1 pathway was detected by RT-PCR and Western blot. Results:After the SW480 cells were treated with 40 and 50 mg·L-1 of DADS for 24 h, the number of transmembrane cells through the Matrigel obviously decreased by 57.12%and 64.59%, respectively (P0.05). After the treatment with 45 mg·L-1 of DADS for 6, 12, 24, and 48 h, the expression of Rac1, Rock1, PAK1, LIMK1, and Destrin proteins respectively decreased in a time-dependent manner compared with the control group (P0.05). Moreover, the expression of p-LIMK1 and p-cofilin1 notably decreased in a time-dependent manner (P<0.05). Conclusion:DADS inhibits cell migration and invasion, which is related to the down-regulation of Rac1, Rock1, PAK1, LIMK1, p-LIMK1, p-cofilin1, and destrin through the Rac1-ADF/cofilin1 pathway.

Relationship between cofilin-1 expression and implantation capacity in eutopic endometrium of patient with endometriosis / 中华妇产科杂志

Objective To investigate the relationship between expression of eofilin-1 and in vivo implantation capacity of eutopic endometrium of endometriosis.Methods Eutopic endometrium of 20 cases with stage Ⅲ or Ⅳ endometriosis were obtained by laparoscopic or laparotomy surgery (endometriosis group) matched with 20 cases of eutopic endometrium from patients with cervical cancer in situ (control group) in Department of Obstetrics and Gynecology,ShengJing Hospital.All cases' eutopic endometrium were collected and injected into abdominal cavity of nude mice to establish endometriosis animal model, then the successful rate of animal model and volume of endometriosis lesion were calculated.The expression and positive rate of eofilin-1 protein were measured by western blot and immunohistochemistry staining.Results The mean volume of endometriosis lesions (2.38 ± 0.22)mm~3 in endometriosis group was significantly bigger than (0.36 ± 0.08) mm~3 in control group (P < 0.05).The successful rate of establishing endometriosis model was 95% (19/20) in endometriosis group and 5% (1/20)in control group, which reached statistical difference(P <0.05).The expression and positive rate of cofilin-1 protein in eutopic endometrium and the expression of cofilin-1 protein in endometriosis lesion of animal model were 0.82±0.06, 90% (18/20), 0.85 ± 0.03 and 0.21 ± 0.03, 20% (4/20), 0.22 ± 0.02 in control group, which reached statistical difference(P <0.05).The successful rate of establishing endometriosis model with positive cofilin-1 in endometrium 86% (19/22) was significantly higher than 6% (1/16) of model's endometrium with negative cofilin-1 expression(P <0.05).Conclusions Implanting capacity and cofilin-1 expression level of eutopic endometrium of endometriosis were more intensive than that of normal endometrium.High cofilin-1 expression was probably related with implanting capacity of eutopic endometrium of endometriosis.