Objective: To investigate the mechanism of S100A7 inducing the migration and invasion in cervical cancers. Methods: Tissue samples of 5 cases of cervical squamous cell carcinoma and 3 cases of adenocarcinoma were collected from May 2007 to December 2007 in the Department of Gynecology of the Affiliated Hospital of Qingdao University. Immunohistochemistry was performed to evaluate the expression of S100A7 in cervical carcinoma tissues. S100A7-overexpressing HeLa and C33A cells were established with lentiviral systems as the experimental group. Immunofluorescence assay was performed to observe the cell morphology. Transwell assay was taken to detect the effect of S100A7-overexpression on the migration and invasion of cervical cancer cells. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to examine the mRNA expressions of E-cadherin, N-cadherin, vimentin and fibronectin. The expression of extracellular S100A7 in conditioned medium of cervical cancer cell was detected by western blot. Conditioned medium was added into Transwell lower compartment to detect cell motility. Exosomes were isolated and extracted from the culture supernatant of cervical cancer cell, the expressions of S100A7, CD81 and TSG101 were detected by western blot. Transwell assay was taken to detect the effect of exosomes on the migration and invasion of cervical cancer cells. Results: S100A7 expression was positively expressed in cervical squamous carcinoma and negative expression in adenocarcinoma. Stable S100A7-overexpressing HeLa and C33A cells were successfully constructed. C33A cells in the experimental group were spindle shaped while those in the control group tended to be polygonal epithelioid cells. The number of S100A7-overexpressed HeLa cells passing through the Transwell membrane assay was increased significantly in migration and invasion assay (152.00±39.22 vs 105.13±15.75, P<0.05; 115.38±34.57 vs 79.50±13.68, P<0.05). RT-qPCR indicated that the mRNA expressions of E-cadherin in S100A7-overexpressed HeLa and C33A cells decreased (P<0.05) while the mRNA expressions of N-cadherin and fibronectin in HeLa cells and fibronectin in C33A cells increased (P<0.05). Western blot showed that extracellular S100A7 was detected in culture supernatant of cervical cancer cells. HeLa cells of the experimental group passing through transwell membrane in migration and invasion assays were increased significantly (192.60±24.41 vs 98.80±47.24, P<0.05; 105.40±27.38 vs 84.50±13.51, P<0.05) when the conditional medium was added into the lower compartment of Transwell. Exosomes from C33A cell culture supernatant were extracted successfully, and S100A7 expression was positive. The number of transmembrane C33A cells incubated with exosomes extracted from cells of the experimental group was increased significantly (251.00±49.82 vs 143.00±30.85, P<0.05; 524.60±52.74 vs 389.00±63.23, P<0.05). Conclusion: S100A7 may promote the migration and invasion of cervical cancer cells by epithelial-mesenchymal transition and exosome secretion.
Female ; Humans ; Uterine Cervical Neoplasms/pathology* ; HeLa Cells ; Fibronectins/metabolism* ; Culture Media, Conditioned ; Carcinoma, Squamous Cell/metabolism* ; Adenocarcinoma ; Cadherins/metabolism* ; RNA, Messenger/metabolism* ; Cell Movement ; Epithelial-Mesenchymal Transition/genetics* ; Cell Line, Tumor ; Cell Proliferation ; S100 Calcium Binding Protein A7/metabolism*

OBJECTIVE: "Multi-targeting" drugs can prove fruitful to combat drug-resistance of multifactorial disease-cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. METHODS: Thuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. RESULTS: Thuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC₅₀) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways-apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC₅₀ of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood-brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. CONCLUSION Robust "multi-targeting" anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.
Animals ; Apoptosis ; Autophagy ; Beclin-1/pharmacology* ; Bicyclic Monoterpenes ; Caspase 3 ; Cell Line, Tumor ; Female ; Humans ; Mammals/metabolism* ; Mice ; Molecular Docking Simulation ; Papillomavirus Infections/drug therapy* ; Phosphatidylinositol 3-Kinases ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use* ; Reactive Oxygen Species/metabolism* ; Thuja/metabolism* ; Uterine Cervical Neoplasms/pathology*

OBJECTIVE: To explore the role of vasohibin-2 (VASH2) in regulation of proliferation and metastasis of cervical cancer cells. METHODS: We analyzed the differentially expressed genes between cervical cancer cells with flotillin-1 overexpression and knockdown by RNA-seq combined with analysis of public databases. The expression levels of VASH2 were examined in normal cervical epithelial cells (HcerEpic), cervical cancer cell lines (HeLa, C-33A, Ca ski, SiHa and MS751) and fresh cervical cancer tissues with different lymph node metastasis status. We further tested the effects of lentivirus-mediated overexpression and interference of VASH2 on proliferation, migration, invasion and lymphatic vessel formation of the cervical cancer cells and detected the expression levels of key epithelial-mesenchymal transition (EMT) markers and TGF-β mRNA. RESULTS: RNA-seq and analysis of public databases showed that VASH2 expression was significantly upregulated in cervical cancer cells exogenously overexpressing flotillin-1 (P < 0.05) and downregulated in cells with flotillin-1 knockdown (P < 0.05), and was significantly higher in cervical cancer tissues with lymph node metastasis than in those without lymph node metastasis (P < 0.01). In cervical cancer cell lines Ca Ski, SiHa, and MS751 and cervical cancer tissue specimens with lymph node metastasis, VASH2 expression was also significantly upregulated as compared with HcerEpic cells and cervical cancer tissues without lymph node metastasis (P < 0.05). Exogenous overexpression of VASH2 significantly promoted proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells, whereas these abilities were significantly inhibited in cells with VASH2 knockdown (P < 0.05). The cervical cancer cells overexpressing VASH2 showed significant down- regulation of e-cadherin and up- regulation of N-cadherin, Vimentin and VEGF-C, while the reverse changes were detected in cells with VASH2 knockdown (P < 0.05). TGF-β mRNA expression was significantly up-regulated in cervical cancer cells overexpressing VASH2 and down-regulated in cells with VASH2 knockdown (P < 0.001). CONCLUSION Flotillin-1 may participate in TGF-β signaling pathway-mediated EMT through its down-stream target gene VASH2 to promote the proliferation, migration, invasion and lymphatic vessel formation of cervical cancer cells in vitro.
Angiogenic Proteins/metabolism* ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation/genetics* ; Epithelial-Mesenchymal Transition/genetics* ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Lymphatic Metastasis ; RNA, Messenger ; Transforming Growth Factor beta/metabolism* ; Uterine Cervical Neoplasms/pathology*

OBJECTIVE: To study the effect of knocking down fascin on cervical cancer cell proliferation and tumorigenicity in nude mice. METHODS: Cervical cancer CaSki cells were infected with a lentiviral vector carrying fascin siRNA or with a negative control lentivirus, and fascin mRNA and protein expressions in the cells were detected using qRT-PCR and Western blotting. MTT assay was used to determine the proliferation of CaSki cells with fascin knockdown. CaSki cells transfected with fascin siRNA or the control lentiviral vector and non-transfected CaSki cells were inoculated subcutaneously in nude mice, and the volume and weight of the transplanted tumor were measured; Western blotting was used to detect the expressions of proliferating cell nuclear antigen (PCNA), survivin, cyclin dependent kinase 4 (CDK4) and p21 proteins in the tumor xenograft. RESULTS: Infection with the lentiviral vector carrying fascin siRNA, but not the negative control vector, caused significant reductions in the expression levels of fascin mRNA and protein in CaSki cells ( < 0.05). Fascin knockdown resulted in significantly reduced proliferation of CaSki cells ( < 0.05). The nude mice inoculated with CaSki cells with fascin knockdown showed reduced tumor volume and weight, lowered levels of PCNA, survivin and CDK4, and increased expression of p21 protein in the tumor xenograft compared with the control mice. The negative control lentivirus did not affect the proliferation or tumorigenicity of CaSki cells in nude mice or the expression levels of PCNA, survivin, CDK4 or p21 proteins in the xenografts. CONCLUSIONS Knocking down fascin can inhibit the growth and tumorigenicity of cervical cancer cells in nude mice.
Animals ; Apoptosis ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase 4 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Female ; Gene Knockdown Techniques ; Genetic Vectors ; Humans ; Mice ; Mice, Nude ; Microfilament Proteins ; genetics ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; Survivin ; metabolism ; Transfection ; Tumor Burden ; Uterine Cervical Neoplasms ; etiology ; pathology

Objective: To explore the relationship between abnormal expression of fragile histidine triad (FHIT) gene and methyl-CpG-binding protein 2 (MeCP2) as well as their interaction on cervical cancerization. Methods: A total of 73 patients with cervical squamous cell carcinoma (SCC), 113 patients with cervical intraepithelial neoplasia (CIN Ⅰ, n=45; CINⅡ/Ⅲ, n=68) and 60 women with normal cervix (NC) were included in the study. Real time PCR and Western blot were performed to detect the expression levels of mRNA and protein about FHIT and MeCP2, respectively. The methylation status of FHIT gene CpG island was tested by methylation-specifc PCR (MSP). Kruskal-Wallis H test, χ(2) test, trend χ(2) test and Spearman correlation analysis were conducted with software SPSS 20.0. The interaction was evaluated by generalized multifactor dimensionality reduction (GMDR) model. Results: With the deterioration of cervical lesion, the methylation rates of FHIT gene CpG island (χ(2)=18.64, P<0.001; trend χ(2)=18.08, P<0.001) increased gradually, while the expression levels of FHIT mRNA (H=27.32, P<0.001; trend χ(2)=12.65, P<0.001) and protein (H=47.10, P<0.001; trend χ(2)=29.79, P<0.001) decreased gradually. There was a negative correlation between the methylation rates of FHIT gene CpG island and the expression level of FHIT protein (r=-0.226, P<0.001). The levels of MeCP2 mRNA (H=26.19, P<0.001; trend χ(2)=11.81, P=0.001) and protein (H=69.02, P<0.001; trend χ(2)=47.44, P<0.001) increased gradually with the aggravation of cervical lesions. There was a positive correlation between the expression level of MeCP2 protein and the FHIT mRNA Ct ratio (r=0.254, P<0.001). Expression of proteins were negatively correlated between MeCP2 and FHIT (r=-0.213, P=0.001). The results analyzed by GMDR model showed that there were interactions among high MeCP2 protein expression, the CpG island methylation of FHIT and mRNA and protein expression in CINⅡ/Ⅲ group, and among high MeCP2 mRNA and protein expression, the CpG island methylation of FHIT and low mRNA and protein expression in SCC group. Conclusion: High expression of MeCP2 mRNA and protein, the CpG island methylation and low mRNA and protein expression of FHIT could increase the risk of cervical carcinogenesis, and there might be a synergistic effect on cervical carcinogenesis.
Acid Anhydride Hydrolases/metabolism* ; Carcinoma, Squamous Cell/pathology* ; DNA Methylation ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Methyl-CpG-Binding Protein 2/metabolism* ; Neoplasm Proteins/metabolism* ; Polymerase Chain Reaction/methods* ; RNA, Messenger ; Uterine Cervical Neoplasms/pathology* ; Uterine Cervical Dysplasia/pathology*

OBJECTIVETo investigate the effect of docosahexaenoic acid (DHA) on apoptosis, migration and invasion of cervical cancer cell lines.

METHODScervical cancer cell lines Hela and Siha in logarithmic phase were treated different concentrations of DHA. The morphological changes of the cells were observed microscopically and cell apoptosis was observed using Hoechst 33258 fluorescent staining. MTT assay was used to evaluate the effect of DHA in suppressing cell growth, and flow cytometry was employed to analyze the changes of cell apoptotic rate following DHA stimulations. Wound healing assay and Transwell migration assay were used to evaluate the migration of the cell lines. The expression levels of Bax, Bcl-2 cleaved caspase3, MMP-9 and VEGF proteins were detected by Western blotting.

RESULTSDHA exposure of the cells caused obvious morphological changes and dose-dependently increased the number of apoptotic bodies in the cells. MTT assay showed that DHA inhibited the growth of the cancer cells in a time- and concentration-dependent manner. DHA also effectively suppressed migration and invasion of the cancer cells. The cells exposed to DHA showed significantly down-regulation of Bcl-2, MMP-9 and VEGF proteins and up-regulation of cleaved-caspase 3 and Bax.

CONCLUSIONDHA can promote cervical carcinoma cell apoptosis by down-regulating the anti-apoptotic proteins Bax, Bcl-2 and cleaved-caspase3 and suppress cell invasion by decreasing MMP-9 and VEGF expressions.

Apoptosis ; Caspase 3 ; metabolism ; Cell Cycle ; Cell Line, Tumor ; drug effects ; Cell Movement ; Cell Proliferation ; Docosahexaenoic Acids ; pharmacology ; Down-Regulation ; Female ; HeLa Cells ; drug effects ; Humans ; Matrix Metalloproteinase 9 ; metabolism ; Neoplasm Invasiveness ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; Vascular Endothelial Growth Factor A ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo investigate the relationship between sensitivity to cisplatin (DDP) and the expression of HSP70 in cervical cancer cells in vitro.

METHODSCervical cancer Hela229 cells treated with different concentrations of DDP and the HSP70 inhibitor (PFT-µ) were examined for cell viability using MTT assay and colony forming ability. The cell apoptosis was analyzed by flow cytometry with propidium iodide staining and DAPI staining, and JC-1 staining was used to determine mitochondrial membrane potential. The expressions of HSP70, Bcl-2, Bax and caspase-3 were measured with Western blotting. A nude mouse model bearing Hela229 cell xenograft was used to evaluate the effect of DDP and PFT-µ on tumor growth.

RESULTSHela229 cells expressed a higher level of HSP70 than normal cervical cells. The combined use of PFT-µ significantly enhanced the inhibitory effect of DDP (P<0.01) and increased the cell apoptosis in Hela229 cells. JC-1 staining demonstrated that DDP combined with PFT-µ more obviously reduced mitochondrial membrane potential. DDP combined with PFT-µ more strongly lowered Bcl-2 expression and increased the expressions of casepase-3 and Bax than DDP alone. In the nude mouse model, PFT-µ significantly enhanced DDP sensitivity of Hela229 cell xenografts (P<0.01).

CONCLUSIONSInhibition of HSP70 expression can enhance the sensitivity of cervical cancer cell to DDP both in vivo and in vitro possibly by promoting cell apoptosis, suggesting the potential of HSP70 as a new target for gene therapy of cervical cancer.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Caspase 3 ; metabolism ; Cell Proliferation ; Cell Survival ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Female ; HSP70 Heat-Shock Proteins ; antagonists & inhibitors ; HeLa Cells ; Humans ; Membrane Potential, Mitochondrial ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Sulfonamides ; pharmacology ; Uterine Cervical Neoplasms ; drug therapy ; pathology ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo explore the effects of silencing HERC4 on the proliferation, apoptosis, and migration of cervical cancer cell line Hela and the possible molecular mechanisms.

METHODSThree HERC4-specific small interfering RNAs (siRNAs) were transfected into Hela cells, and HERC4 expression in the cells was examined with Western blotting. CCK-8 assay, annexin V-FITC/PI assay, and wound healing assay were used to assess the effect of HERC4 silencing on the proliferation, apoptosis and migration ability of Hela cells. The expression levels of cyclin D1 and Bcl-2 in the cells were detected using Western blotting.

RESULTSTransfection of siRNA-3 resulted in significantly decreased HERC4 protein expression (P<0.01). HERC4 silencing by siRNA-3 markedly suppressed the proliferation and migration of Hela cells, increased the apoptosis rate (P<0.01) and reduced the expression levels of cyclin D1 and Bcl-2 (P<0.01).

CONCLUSIONSilencing of HERC4 efficiently inhibits the proliferation, migration, and invasion of Hela cells in vitro, and the underlying mechanisms may involve the down-regulation of cyclin D1 and Bcl-2.

Apoptosis ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin D1 ; metabolism ; Down-Regulation ; Female ; HeLa Cells ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; Ubiquitin-Protein Ligases ; genetics ; metabolism ; Uterine Cervical Neoplasms ; pathology

OBJECTIVE: We investigated the prognostic value of intratumoral [18F]fluorodeoxyglucose (FDG) uptake heterogeneity (IFH) derived from positron emission tomography/computed tomography (PET/CT) in patients with cervical cancer. METHODS: Patients with uterine cervical cancer of the International Federation of Obstetrics and Gynecology (FIGO) stage IB to IIA were imaged with [18F]FDG PET/CT before radical surgery. PET/CT parameters such as maximum and average standardized uptake values (SUV(max) and SUV(avg)), metabolic tumor volume (MTV), total lesion glycolysis (TLG), and IFH were assessed. Regression analyses were used to identify clinicopathological and imaging variables associated with progression-free survival (PFS). RESULTS: We retrospectively reviewed clinical data of 85 eligible patients. Median PFS was 32 months (range, 6 to 83 months), with recurrence observed in 14 patients (16.5%). IFH at an SUV of 2.0 was correlated with primary tumor size (p<0.001), SUV(tumor) (p<0.001), MTV(tumor) (p<0.001), TLG(tumor) (p<0.001), depth of cervical invasion (p<0.001), and negatively correlated with age (p=0.036). Tumor recurrence was significantly associated with TLG(tumor) (p<0.001), MTV(tumor) (p=0.001), SUV(LN) (p=0.004), IFH (p=0.005), SUV(tumor) (p=0.015), and FIGO stage (p=0.015). Multivariate analysis identified that IFH (p=0.028; hazard ratio, 756.997; 95% CI, 2.047 to 279,923.191) was the only independent risk factor for recurrence. The Kaplan-Meier survival graphs showed that PFS significantly differed in groups categorized based on IFH (p=0.013, log-rank test). CONCLUSION: Preoperative IFH was significantly associated with cervical cancer recurrence. [18F]FDG based heterogeneity may be a useful and potential predicator of patient recurrence before treatment.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell/metabolism/*radionuclide imaging/secondary ; Disease-Free Survival ; Female ; Fluorodeoxyglucose F18/*pharmacokinetics ; Glycolysis ; Humans ; Middle Aged ; Multimodal Imaging ; Neoplasm Invasiveness ; Neoplasm Recurrence, Local/metabolism/radionuclide imaging ; Neoplasm Staging ; *Positron-Emission Tomography ; Predictive Value of Tests ; Radiopharmaceuticals/*pharmacokinetics ; Retrospective Studies ; Tomography, X-Ray Computed ; Tumor Burden ; Uterine Cervical Neoplasms/metabolism/pathology/*radionuclide imaging

OBJECTIVE: To investigate the effects of 7-difluoromethy-5, 4'- dimethoxygenistein (DFMG) on inhibiting proliferation and inducing apoptosis of human cervical cancer HeLa cells and its possible molecular mechanism in vitro.
 METHODS: HeLa cells were cultured in vitro. The effect of DFMG on inhibiting proliferation was determined using MTT assay. The effects of DFMG on inducing apoptosis were assessed using flow cytometry with AV-PI staining, AO/EB staining, and agarose gel electrophoresis. Multiple molecular techniques, such as RT-PCR, Western blot, siRNA transfection, and cDNA transfection, were used to explore its possible molecular mechanism.
 RESULTS: DFMG presented with dramatically inhibiting proliferation effect of HeLa cells in a time-and dose-dependent manner ranging from 0.25 to 64 μg/mL and from 24 to 72 h in vitro, and its IC(50) was 4.62 μg/mL for 48 h. The cells treated with DFMG for 48 h showed typical morphological change of apoptosis, typical DNA ladder of agarose gel electrophoresis, and the sub-G(1) population increased in a dose-dependent manner. Simultaneously the expressions of c-myc mRNA, c-myc protein and its downstream genes, such as bax, cyto-c and caspase-9, were up-regulated, while bcl-2 protein was down-regulated. Down-regulation of c-myc by siRNA attenuated DFMG-induced cell proliferation inhibition and inducing apoptosis. Up-regulation expression of c-myc by cDNA transfection could enhance the effects of DFMG-induced cell proliferation inhibition and inducing apoptosis.
 CONCLUSION DFMG could inhibit the proliferation and induce the apoptosis of human cervical cancer HeLa cells in vitro, and its mechanism may be closely related to regulate c-myc and its down-stream gene expression.
Apoptosis ; drug effects ; Caspase 9 ; metabolism ; Cell Proliferation ; drug effects ; Down-Regulation ; Female ; Gene Expression Regulation, Neoplastic ; Genistein ; analogs & derivatives ; pharmacology ; HeLa Cells ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Proto-Oncogene Proteins c-myc ; metabolism ; Up-Regulation ; Uterine Cervical Neoplasms ; pathology ; bcl-2-Associated X Protein ; metabolism