Primary Treatment for Clinically Early Cervical Cancer with Lymph Node Metastasis: Radical Surgery or Radiation?

OBJECTIVE: To compare survival outcomes between primary radical surgery and primary radiation in early cervical cancer. METHODS: Patient information was extracted from the Surveillance, Epidemiology, and Results database. Patients diagnosed with early cervical cancer of stage T1a, T1b, and T2a (American Joint Committee on Cancer, 7th edition) from 1998 to 2015 were included in this study after propensity score matching. Overall survival (OS) was analyzed using the Kaplan-Meier method. RESULTS: Among the 4964 patients included in the study, 1080 patients were identified as having positive lymph nodes (N1), and 3884 patients were identified as having negative lymph nodes (N0). Patients with primary surgery had significantly longer 5-year OS than those with primary radiotherapy in both the N1 group (P<0.001) and N0 group (P<0.001). In the subgroup analysis, similar results were found in patients with positive lymph nodes of stage T1a (100.0% vs. 61.1%), T1b (84.1% vs. 64.3%), and T2a (74.4% vs. 63.8%). In patients with T1b1 and T2a1, primary surgery resulted in longer OS than primary radiation, but not in patients with T1b2 and T2a2. In multivariate analysis, the primary treatment was identified as an independent prognostic factor in both N1 and N0 patients (HRN1=2.522, 95% CI=1.919-3.054, PN1<0.001; HRN0=1.895, 95% CI=1.689-2.126, PN0<0.001). CONCLUSION: In early cervical cancer stage T1a, T1b1, and T2a1, primary surgery may result in longer OS than primary radiation for patients with and without lymph node metastasis.

Adjuvant Chemotherapy versus Radiotherapy in High-risk, Early-stage Endometrioid Endometrial Carcinoma.

OBJECTIVE: The present study was designed to evaluate the effects of adjuvant chemotherapy (CT) vs. radiotherapy (RT, alone or combined with CT) on the prognosis of patients with high-risk, early-stage (stage I and stage II) endometrioid endometrial carcinoma. METHODS: This single-center retrospective clinical study was conducted in Union Hospital, Tongji Medical College, Huazhong University of Science and Technology between 2010 and 2019. In the present study, endometrioid endometrial carcinoma patients, who underwent total hysterectomy and bilateral salpingo-oophorectomy followed by postoperative adjuvant CT or RT (alone or combined with CT), and were diagnosed with stage IA grade 2/3 with lymph-vascular space invasion (LVSI), and stage IB with two or more uterine risks, including old age, histological grade 2 or 3, LVSI and stage II, were included. According to the postoperative adjuvant therapy, all eligible patients were divided into two groups: CT group and RT (RT±CT) group. The primary objective was to investigate overall survival (OS) and disease-free survival (DFS) between the CT and RT groups. Grade 3 or worse adverse events were also presented in the present study. RESULTS: A total of 145 eligible patients were included. Among these patients, 97 patients underwent adjuvant CT and 48 patients underwent adjuvant RT (RT±CT). The median follow-up was 47.2 months, and the five-year OS rate was 92.7% in the CT group and 88.6 % in the RT group [hazard ratio (HR): 0.81, 95% confidence interval (CI): 0.22-2.99). The 5-year DFS rate for the two groups was 85.7% and 80.2%, respectively (HR: 0.82, 95% CI: 0.33-2.05). The cumulative incidence of local-regional disease recurrence at 60 months of follow-up was 6.2% in the CT group and 6.3% in the RT group (HR=1.11; 95%CI: 0.28-4.35). The cumulative incidence of distant recurrence at 60 months of follow-up was 5.2% in the CT group and 10.4% in the RT group (HR=0.65; 95%CI: 0.19-2.24). Both groups of patients were well-tolerant, and the only grade 3 or worse adverse events were neutropenia and thrombocytopenia. CONCLUSION: There was no difference in efficacy for adjuvant CT or adjuvant RT (RT±CT) in high-risk, early-stage endometrioid endometrial carcinoma. CT exhibited a trend of reducing the distant relapse, although there was no significant difference, when compared with adjuvant RT (RT±CT).

Meloidogyne graminicola protein disulfide isomerase may be a nematode effector and is involved in protection against oxidative damage.

The rice root-knot nematode, Meloidogyne graminicola, is a serious pest in most rice-growing countries. Usually, nematodes employ antioxidants to counteract the harm of reactive oxygen species (ROS) and facilitate their infection. Here the gene encoding M. graminicola protein disulphide isomerase (MgPDI) was identified. The deduced protein is highly conserved in the putative active-site Cys-Gly-His-Cys. In situ hybridization showed that MgPDI was specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2s). MgPDI was significantly up-regulated in the late parasitic J2s. Characterization of the recombinant protein showed that the purified MgPDI exhibited similar activities to other oxidases/isomerases such as the refolding of the scrambled RNase and insulin disulfide reductase and the protection of plasmid DNA and living cells from ROS damage. In addition, silencing of MgPDI by RNA interference in the pre-parasitic J2s lowered their multiplication factor. MgPDI expression was up-regulated in the presence of exogenous H2O2, whereas MgPDI silencing resulted in an increase in mortality under H2O2 stress. MgPDI is localized in the apoplast when transient expression in Nicotiana benthamiana leaves. The results indicated that MgPDI plays important roles in the reproduction and pathogenicity of M. graminicola and it also contributes to protecting nematodes from exogenous H2O2 stress.

Mitochondria-Accumulating Rhenium(I) Tricarbonyl Complexes Induce Cell Death via Irreversible Oxidative Stress and Glutathione Metabolism Disturbance.

Mitochondria play a critical role in tumorigenesis. Targeting mitochondria and disturbing related events have been emerging as a promising way for chemotherapy. In this work, two binuclear rhenium(I) tricarbonyl complexes of the general formula [Re2(CO)6(dip)2L](PF6)2 (dip = 4,7-diphenyl-1,10-phenanthroline; L = 4,4'-azopyridine (ReN) or 4,4'-dithiodipyridine (ReS)) were synthesized and characterized. ReN and ReS can react with glutathione (GSH). They exhibit good in vitro anticancer activity against cancer cell lines screened. Besides, they can target mitochondria, cause oxidative stress, and disturb GSH metabolism. Both ReN and ReS can induce necroptosis and caspase-dependent apoptosis simultaneously. We also demonstrate that ReN and ReS can inhibit tumor growth in nude mice bearing carcinoma xenografts. Our study shows the potential of Re(I) complexes as chemotherapeutic agents to kill cancer cells via a mitochondria-to-cellular redox strategy.

Toxicity of seven insecticides to different developmental stages of the whitefly Bemisia tabaci MED (Hemiptera: Aleyrodidae) in multiple field populations of China.

Chemical control is important in the management of the tobacco whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Susceptibility of B. tabaci to insecticides may vary among different developmental stages and geographical populations. In this study, we examined toxicity of seven commonly-used insecticides to B. tabaci MED in four field populations from China. Avermectin has high level of toxicity to all stages of B. tabaci MED in all four populations. Cyantraniliprole and sulfoxaflor have high toxicity to adults. Spirotetramat, cyantraniliprole and flonicamid have high toxicity to nymphs but not adults. Acetamiprid, cyantraniliprole and sulfoxaflor have high toxicity to eggs. However, the relative toxicity of B. tabaci MED to these chemicals varied across different populations, with little consistency in population differences across developmental stages. Our findings together with some instances where LC95 values were higher than field recommended dosages indicate field-evolved resistance to insecticides (such as thiamethoxam and sulfoxaflor) and stage-specific mechanisms that will influence effective control of B. tabaci MED by insecticides.

Repeated inhalation of sevoflurane inhibits the information transmission of Purkinje cells and delays motor development via the GABAA receptor ε subunit in neonatal mice.

General anesthesia is widely used in pediatric surgery, although the influence of general anesthesia on cerebellar information transmission and motor function is unclear. In the present study, neonatal mice received repeated inhalation of sevoflurane, and electrophysiological alterations in Purkinje cells (PCs) and the development of motor functions were detected. In addition, γ­aminobutyric acidA receptor ε (GABAA­R ε) subunit knockout mice were used to investigate the mechanism of action of sevoflurane on cerebellar function. In the neonatal mice, the field potential response of PCs induced by sensory stimulation and the motor function indices were markedly inhibited by sevoflurane, and the inhibitory effect was positively associated with the number of repetitions of anesthesia. In additional the GABAA­R ε subunit level of PCs was promoted by sevoflurane in a dose­dependent manner, and the inhibitory effects of sevoflurane on PC field potential response and motor function were alleviated in GABAA­R ε subunit knockout mice. The GABAA­R ε subunit was activated by sevoflurane, leading to inhibition of sensory information transmission in the cerebellar cortex, field potential responses of PCs and the development of cerebellar motor function. The present study provided experimental evidence for the safe usage of sevoflurane in clinical anesthesia, and suggested that GABAA­R ε subunit antagonists may be considered for combined application with general anesthesia with repeated inhalation of sevoflurane, for adverse effect prevention in the clinic.

Research Progress of MicroRNA in Early Detection of Ovarian Cancer.

OBJECTIVE: This review aimed to update the progress of microRNA (miRNA) in early detection of ovarian cancer. We discussed the current clinical diagnosis methods and biomarkers of ovarian cancer, especially the methods of miRNA in early detection of ovarian cancer. DATA SOURCES: We collected all relevant studies about miRNA and ovarian cancer in PubMed and CNKI from 1995 to 2015. STUDY SELECTION: We included all relevant studies concerning miRNA in early detection of ovarian cancer, and excluded the duplicated articles. RESULTS: miRNAs play a key role in various biological processes of ovarian cancer, such as development, proliferation, differentiation, apoptosis and metastasis, and these phenomena appear in the early-stage. Therefore, miRNA can be used as a new biomarker for early diagnosis of ovarian cancer, intervention on miRNA expression of known target genes, and potential target genes can achieve the effect of early prevention. With the development of nanoscience and technology, analysis methods of miRNA are also quickly developed, which may provide better characterization of early detection of ovarian cancer. CONCLUSIONS: In the near future, miRNA therapy could be a powerful tool for ovarian cancer prevention and treatment, and combining with the new analysis technology and new nanomaterials, point-of-care tests for miRNA with high throughput, high sensitivity, and strong specificity are developed to achieve the application of diagnostic kits in screening of early ovarian cancer.

Establishing a Nomogram for Stage IA-IIB Cervical Cancer Patients after Complete Resection.

BACKGROUND: This study aimed to establish a nomogram by combining clinicopathologic factors with overall survival of stage IA-IIB cervical cancer patients after complete resection with pelvic lymphadenectomy. MATERIALS AND METHODS: This nomogram was based on a retrospective study on 1,563 stage IA-IIB cervical cancer patients who underwent complete resection and lymphadenectomy from 2002 to 2008. The nomogram was constructed based on multivariate analysis using Cox proportional hazard regression. The accuracy and discriminative ability of the nomogram were measured by concordance index (C-index) and calibration curve. RESULTS: Multivariate analysis identified lymph node metastasis (LNM), lymph-vascular space invasion (LVSI), stromal invasion, parametrial invasion, tumor diameter and histology as independent prognostic factors associated with cervical cancer survival. These factors were selected for construction of the nomogram. The C-index of the nomogram was 0.71 (95% CI, 0.65 to 0.77), and calibration of the nomogram showed good agreement between the 5-year predicted survival and the actual observation. CONCLUSIONS: We developed a nomogram predicting 5-year overall survival of surgically treated stage IA-IIB cervical cancer patients. More comprehensive information that is provided by this nomogram could provide further insight into personalized therapy selection.

[Evaluation the efficacy and safety of estradiol and drospirenone tablets in the treatment of menopausal symptoms among postmenopausal Chinese healthy women:a randomized, multi-center, double-blind, placebo-controlled clinical study].

OBJECTIVE: To study the efficacy and safety of estradiol and drospirenone tablets (Angeliq) in treatment of menopausal symptoms among postmenopausal Chinese healthy women. METHODS: Total 244 postmenopausal Chinese healthy women who had moderate to severe hot flushes were randomly assigned into estradiol and drospirenone (observation group, n = 183) or placebo group (n = 61) by the ratio of 3:1 for 16 weeks in this randomized multi-center double-blind placebo-controlled study. During the trial, the follow-up visits were conducted at week 4, 8, 12, 16 of treatment and 2 weeks after treatment respectively. Height, weight, vital signs, hot flushes, other relevant menopausal symptoms and vaginal bleeding were observed in each follow-up visit, while the clinical global impression scale was assessed at 16 weeks as well. RESULTS: It showed that hot flushes were reduced significantly more in observation group than that in placebo group (P < 0.01), although both treatments were effective. The absolute values of mean severity index of total hot flushes decreased by -0.6 ± 0.5 in observation group and -0.4 ± 0.4 in placebo group from baseline respectively, which reached significant difference (P < 0.05). However, the absolute values of mean severity index of moderate to severe hot flushes decreased by -0.6 ± 0.8 in observation group and -0.3 ± 0.6 in placebo group from baseline respectively, which had no significant difference (P > 0.05). After 16 weeks treatment, it also showed that estradiol and drospirenone had significant better efficacy than placebo on moderate to severe sweating, vaginal dryness and clinical global impression scale (P < 0.01). During the trial, blood pressure in observation group was stable. The rate of vaginal bleeding in observation group was higher than that in the placebo group, especially during the week 4 to week 8 when 48.9% (87/178) in observation group and 10.7% (6/56) in placebo group of patients bled. Although the cumulative amenorrhea rate of observation group was lower than that of placebo group in each cycle (28 days), it increased gradually along with duration of the treatment. The commonest adverse event in observation group was breast tenderness which accounted for 12.0% (22/183). The level of serum potassium was in the normal range in observation group mostly.Meanwhile, the other adverse events rate was low. Serious adverse events reported in this trial were assessed as not study drug related or as unlikely study drug related. CONCLUSION: Estradiol and drospirenone tablets which could effectively alleviate menopausal symptoms in postmenopausal Chinese healthy women is a novel hormone replacement therapy regimen with high safety and efficacy.

Influence of deleted in colorectal carcinoma gene on proliferation of ovarian cancer cell line SKOV-3 in vivo and in vitro.

OBJECTIVE: To elucidate the effects of the deleted in colorectal carcinoma (DCC) gene on proliferation of ovarian cancer cell line SKOV-3. METHOD: An exogenous recombinant eukaryotic expression vector pcDNA3.1(+)-DCC, containing human DCC cDNA coding sequences, was constructed and transfected into SKOV-3 cells (SKOV-3/DCC). The pcDNA3.1 (+) transfected cells (SKOV-3/Neo) and SKOV-3 cells were used as the positive and negative controls, respectively. Expressions of DCC mRNA and protein were analyzed by RT-PCR and immunocytochemical analysis, respectively. Cell growth was detected by soft agar colony formation assay and MTT assay. Flow cytometry and transmission electron microscopy were used to assess the effects of DCC on cell cycle distribution and ultrastructure, respectively. BALB/c mice were used to evaluate the effects of DCC on tumorigenicity in vivo. RESULTS: RT-PCR and immunocytochemical analysis revealed the exogenous DCC gene was successfully transfected into SKOV-3 cell lines and obtained permanent expression. The half maximal inhibitory concentration (IC50) of SKOV-3/DCC cells was significantly lower than that of SKOV-3 or SKOV-3/Neo cells (all P<0.05). DCC expression caused SKOV-3 cells to be arrested in G1 phase (78.0%), and electron microscopic analysis showed SKOV-3/DCC cells displayed typical morphological changes of apoptosis. Two mice xenografted with SKOV-3/DCC cells showed no tumor tumorigenecity. The tumor volume of BALB/c mice bearing SKOV-3/DCC cells (3.403 mm(3)) was smaller than that of SKOV-3 cells (9.206 mm(3)). CONCLUSION: DCC gene may play an important role in suppressing the growth of SKOV-3 cell line and inducing apoptosis.

Up-regulation of human arrest-defective 1 protein is correlated with metastatic phenotype and poor prognosis in breast cancer.

BACKGROUND: Human arrest defective 1 protein (ARD1), as a N-terminal acetyltransferase, has been reported to play a crucial role in tumorigenesis, but the results are somewhat controversial. To explore the clinical and pathological significance of ARD1 in breast tumorigenesis, we analyzed ARD1 status in multiple types of breast disease. METHODS: The expression of ARD1 protein was assessed by immunohistochemistry in 356 cases including 82 invasive ductal carcinomas (IDC), 159 fibroadenomas, 66 hyperplasia of mammary glands, 19 inflammatory breast disease, 30 breast cysts, and in 29 postoperative treatment patients. We assessed the relationship of ARD1 protein with clinical and pathological characteristics using χ2 test. RESULTS: ARD1 protein was observed at 61.0% (50/82), 54.7% (87/159), 37.9% (25/66), 36.8% (7/19) in IDC, fibroadenoma, hyperplasia, and inflammation, respectively, and less than 30.0% for breast cyst. Thus, high ARD1 expression correlated with breast cancer (relative risk = 1.32, P < 0.005). Moreover, the level of ARD1 protein in carcinoma patients was distinctly related to lymph node metastasis and ER status, with 94.0% (47/50) as copmpared to 6.0% (3/50) in metastatic and non-metastatic (P < 0.001), and 84.0% (42/50) and 16.0% (8/50) for ER + and ER - (P < 0.01), respectively. In addition, the level of ARD1 appeared to have potential for evaluation of prognosis in breast cancer patients after postoperative therapy. CONCLUSIONS: These results suggest that ARD1 expression may be as a potential target for exploring the mechanism of breast cancer metastasic to lymph nodes and hormone-responsive regulation.

[Role of a latent soluble TNF receptor type I (hsTNFRI) fusion protein in targeted treatment of endometriosis].

OBJECTIVE: To construct a latent human soluble tumor necrosis factor receptor I (hsTNFRI) using the latency associated protein (LAP) of transforming growth factor-beta1 (TGF-beta1) fused via a matrix metalloproteinase (MMP) cleavage site to hsTNFRI so as to detect the latent biological activity of LAP-MMP-hsTNFRI fusion protein. METHODS: A double-stranded deoxyoligonucleotide coding for MMP cleavage site was cloned into plasmid pcDNA3.1. LAP and hsTNFRI cDNA were then inserted into both two sides of MMP cleavage site. After being transferred by LAP-MMP-hsTNFRI fusion gene with liposome, the expression of fusion protein in COS-7 cells was detected by RT-PCR and Western blot. The inhibitory effect of fusion protein upon cytotoxicity of TNF-alpha was detected by methyl thiazolyl tetrazolium (MTT) assay before and after the fusion protein incubated in MMP or peritoneal fluid from endometriosis patients. RESULTS: The recombinant plasmid LAP-MMP-hsTNFRI-pcDNA3.1 was constructed successfully and was expressed effectively in COS-7 cells. The MTT assay showed that there was no difference in the mortality rate of L929 cells between LAP-MMP-hsTNFRI-pcDNA3.1 and empty vector transfection groups (P > 0.05). The mortality rates of L929 cells with 800 ng/L TNF-alpha in LAP-MMP-hsTNFRI-pcDNA3.1 transfection group after incubation with MMP or peritoneal fluid from endometriosis patients were (44.5 +/- 2.4)% and (33.8 +/- 1.9)% respectively. And it was lower than the pre-incubation period (58.1 +/- 2.4)% (P < 0.05). CONCLUSION: The biological activity of LAP-MMP-hsTNFRI fusion protein can be made latent by LAP and activated by peritoneal fluid from endometriosis. Thus a new method has been provided for a targeted therapy of endometriosis.

[Expression of microRNA 27a and its correlation with drug resistance in human ovarian cancer A2780/Taxol cells].

OBJECTIVE: To investigate the expression of microRNA 27a (miR-27a) and relationship with drug resistance in human ovarian cancer A2780/Taxol cells. METHODS: A stem-loop-mediated real-time PCR was used to detect miR-27a expression in A2780 and A2780/Taxol cells. The cells were transfected with the mimics or inhibitors of miR-27a or negative control RNA (NC) by lipofectamine 2000. The expressions of MDR1 gene, P-glycoprotein (P-gp) and homeodomain-interacting protein kinase 2 (HIPK2) protein levels were measured by real-time PCR and western blot respectively. Methyl thiazolyl tetrazolium (MTT) assay was used to analyze drug sensitivity. Apoptosis analysis was measured by fluorescence activated cell sorter (FACS). RESULTS: (1) miR-27a was an average of 2.2-fold higher expression level in A2780/Taxol cells than that in A2780 cells, with a significant difference between the two groups (P<0.05). (2) A2780/Taxol cells transfection with inhibitors of miR-27a showed that the levels of MDR1 mRNA was decreased by 39%, P-gp protein level [(26+/-5)%] decreased than that in the NC group [(43+/-7)%], HIPK2 protein level [(30+/-6)%] increased than that in the NC group [(19+/-4)%], the 50% inhibition concentration (0.5 micromol/L) was less than that in the NC group (6.8 micromol/L), apoptosis rate [(32.5+/-3.6)%] was higher than that in the NC group [(5.6+/-2.1)%], and there were significant differences between two groups (all P<0.05). (3) Transfection of A2780 cells with mimics of miR-27a led to increase MDR1 mRNA expression by 121% as compared with one transfection with NC (P<0.05). CONCLUSION: The expression of miR-27a is upregulated in A2780/Taxol cells, which may regulate MDR1 and P-gp expression by targeting HIPK2.

Stabilization of mismatch repair gene PMS2 by glycogen synthase kinase 3beta is implicated in the treatment of cervical carcinoma.

BACKGROUND: PMS2 expression loss was reported in a variety of human. However, its importance has not been fully understood in cervical carcinoma. The aim of this study was to determine the expression of PMS2 in cervical carcinoma and evaluate the significance of mismatch repair gene PMS2 regulated by glycogen synthase kinase 3beta (GSK-3beta) in chemosensitivity. METHODS: We examined PMS2 and phosphorylated GSK-3beta(s9) expression in cervical carcinoma tissues using immunohistochemical staining. Furthermore, we detected PMS2 expression in HeLa cells and evaluate the interaction with GSK-3beta after transfection with GSK-3beta by small interference RNA (siRNA), co-immunoprecipitation and immunoblotting. We also evaluated the effect of PMS2 transfection on HeLa cells' chemosensitivity to cisplatin treatment. RESULTS: We found significant downregulation of PMS2 in cervical carcinoma, which was negatively associated with phosphorylated GSK-3beta (s9). Furthermore, we demonstrated GSK-3beta transfection was able to interact with PMS2 and enhance PMS2 production in HeLa cells, and increased PMS2 production was responsible for enhanced chemosensitivity. CONCLUSIONS: Our results provide the evidence that stabilization of PMS2 production by GSK-3beta was important to improve chemosensitization, indicating the significance of GSK-3beta-related PMS2 downregulation in the development of cervical carcinoma and in developing a potential strategy for chemotherapy.

[Effects of tumor metastasis suppressor gene nm23-H1 on invasion and proliferation of cervical cancer cell lines].

BACKGROUND AND OBJECTIVE: Previous studies have shown that nm23-H1 is a tumor metastasis suppressor gene. Nucleotide diphosphate kinase 1 (NDPK1) encoded by nm23-H1 is involved in cancer cellular differentiation, proliferation, apoptosis and metastasis. This study was to investigate the effects of nm23-H1 on proliferation and invasion of cervical cancer cells. METHODS: The eukaryotic expression vector pcDNA3.1-nm23-H1 was transfected into cervical cancer cells. Cell invasion potential was determined by the Transwell assay. Cell proliferation was measured by MTT assay and changes in cell cycle distribution were analyzed by flow cytometry (FCM). RESULTS: Compared with parent cells (Caski and SiHa) and vector control cells (Caski-3.1 and SiHa-3.1), the proliferation and invasion of pcDNA3.1-nm23-H1 transfected cells (SiHa-N and Caski-N) were apparently decreased (P<0.05); the proportions of G2/M and S cells were obviously decreased while that of G0/G1 cells was increased (P<0.05). However, transfection of nm23-H1 gene had no influence on proliferation, cell cycle and invasion of HeLa cells (P>0.05). CONCLUSION: nm23-H1 gene could inhibit proliferation and invasion of cervical cancer cells in a cell-dependent manner.

[Roles of vascular endothelial growth factor and platelet-derived growth factor in lymphangiogenesis in epithelial ovarian carcinoma.].

OBJECTIVE: To assess roles of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in the mechanisms of lymphangiogenesis in epithelial ovarian carcinoma. METHODS: (1) Expression of Prox1, a newly described lymphatic endothelial cell nucleus marker, VEGF-A, VEGF-C, VEGF-D and PDGF-A, PDGF-B, PDGF-C, PDGF-D were detected by RT-PCR in SKOV3 cell line and in 90 ovarian tissue samples, included 15 benign tumors, 10 borderline tumors, 45 malignant tumors and 20 normal ovarian samples. (2) Expression levels of Prox1, VEGF-A, -C, -D and PDGF-A, -B, -C, -D were detected in 90 ovarian tissue sample mentioned above by real-time quantitative PCR (RTQ-PCR). RESULTS: (1) Prox1 was expressed in ovarian samples mentioned above, while not detected in SKOV3 cell. VEGF-A, -C, -D and PDGF-A, -B, -C, -D were found in SKOV3 cell and various ovarian tissues. (2) Expression levels of Prox1 (2.2 +/- 1.3, P < 0.01), VEGF-A (3.5 +/- 1.5, P < 0.01), VEGF-C (19 +/- 14, P < 0.01), VEGF-D (3.0 +/- 1.8, P < 0.01) and PDGF-A (3.3 +/- 3.3, P < 0.05), PDGF-C (6.9 +/- 4.6, P < 0.01) in malignant group were found to be significantly higher than those in borderline group and benign group. (3) The expression levels of Prox1, VEGF-A and PDGF-A were significantly greater in samples from the patients with lymph node metastasis (Prox1: 3.0 +/- 1.4, VEGF-A: 4.1 +/- 1.7, PDGF-A: 4.9 +/- 4.1), peritoneum metastasis (Prox1: 2.8 +/- 0.9, VEGF-A: 4.0 +/- 1.8, PDGF-A: 4.5 +/- 4.0) and in stage III - IV (Prox1: 2.6 +/- 1.3, VEGF-A: 4.0 +/- 1.4, PDGF-A: 4.1 +/- 3.7) than those without lymph node metastasis, without peritoneum metastasis and in stage I - II. There was a significant increased in the degree of VEGF-C and VEGF-D expression in positive lymph node metastasis group (VEGF-C: 24 +/- 13, VEGF-D: 3.9 +/- 2.0) compared with negative group (P < 0.05). (4) There were significant positive correlations between the expression levels of Prox1 and VEGF-D (r = 0.62, P < 0.01), PDGF-C (r = 0.91, P < 0.01) or PDGF-D (r = 0.61, P < 0.01). CONCLUSIONS: VEGF-A, VEGF-C and PDGF-A may promote lymphatic metastasis in epithelial ovarian carcinoma through else mechanisms other than lymphangiogenesis. VEGF-D may facilitate lymphangiogenesis and lymph node metastasis in epithelial ovarian cancer. There is no significant correlation between the expression of PDGF-B and lymphangiogenesis and lymph node metastasis. PCGF-C and PDGF-D may motivate lymphangiogenesis, but could not participate in lymph node metastasis in ovarian carcinoma.

[The role and mechanism of CXCR4 and its ligand SDF-1 in the development of cervical cancer metastasis].

BACKGROUND & OBJECTIVE: The chemokine receptor CXCR4 and its sole ligand stromal cell-derived factor-1 (SDF-1) not only actively participate in inflammation, hematopoiesis, infection of HIV, but also play a pivotal role in migration, invasion and metastasis of some malignant tumors. This study was to investigate the role of CXCR4/SDF-1 axis in mediating metastasis in cervical cancer cells through activating the mitogen-activated protein kinase (MAPK) pathway and its possible mechanism. METHODS: Intracellular calcium mobilization was observed under laser scanning confocal fluorescence microscopy. The phospharylation of extracellular signal-regulated kinase (ERK) 1/2 in HeLa cells after binding of SDF-1alpha to CXCR4 was measured by Western blot. Adhesion of CXCR4/SDF-1 to cervical cancer cells and secretion of matrix metalloproteinase (MMP) were detected by adhesion assay and gelatin zymography, respectively. RESULTS: After SDF-1alpha was bound to CXCR4, a rapid and robust mobilization of intracellular calcium in Hela cells was initiated. The difference between the average baseline fluorescence intensity (FI) and the peak FI was significant (P < 0.01). ERK-1/2 was rapidly phosphorylated in Hela cells after its exposure to SDF-1alpha, and the strongest phosphorylation occurred at 30 min. The adhesion ability of Hela cells to fibronectin (FN) and laminin (LN) was increased after SDF-1alpha treatment (P < 0.05 and P < 0.01, respectively), while pretreatment of Hela cells with an ERK-1/2 inhibitor, PD98059, decreased adhesion of Hela cells to extracellular matrix (ECM) with the presence of SDF-1alpha (P < 0.05). Increased amounts of active MMP-2 were secreted in response to increased SDF-1alpha concentrations. When the concentration of SDF-1alpha was 800ng/mL, the secretion of active MMP-2 reached the peak and started to decrease afterwards. CONCLUSION: CXCR4/SDF-1 participates in tumor invasiveness and metastasis in cervical cancer through regulating the adhesion ability by activating the MAPK signaling transduction pathway and promoting secretion of MMP-2.

[Relationship between RAR-beta gene expression defect and its methylation].

OBJECTIVE: To evaluate the expression of RAR-beta gene in cervical carcinoma cell lines SiHa, HeLa, C33A and Caski and to analyze the relation between their gene expression and the promoter methylation of RAR-beta DNA. METHODS: The expression of mRNA and protein of RAR-beta gene in the four cell lines were analyzed by RT-PCR, western blot and immunofluorescence, respectively. Methylation specific PCR (MSP) was used to check whether there was methylation in the promoter of RAR-beta gene. The demethylating agent 5-aza-2'-deoxycytidine (5-Aza-cdR) was used to treat methylated cell lines and the change of RAR-beta gene methylation and RAR-beta gene expression defects were observed. The cell proliferation was assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide method. RESULTS: The mRNA and protein expression levels of RAR-beta in cell lines SiHa, HeLa, Caski and C33A were 0.25 +/- 0.08, 0, 0.60 +/- 0.19, 3.12 +/- 0.92 and 0.23 +/- 0.07, 0, 0.14 +/- 0.05, 0.68 +/- 0.21, respectively. The mRNA and protein expression of RAR-beta in SiHa, HeLa and Caski cell lines were decreased or silenced, whereas its expression increased in C33A cell line. MSP method showed that there were RAR-beta gene methylation in SiHa, HeLa and Caski cell lines, while there was no RAR-beta gene methylation in C33A cell line. After treated with 5-Aza-cdR, the mRNA and protein expression levels of RAR-beta in SiHa, HeLa, Caski and C33A cell lines were 1.82 +/- 0.59, 2.13 +/- 0.62, 1.67 +/- 0.43, 2.95 +/- 0.89 and 0.69 +/- 0.21, 0.83 +/- 0.29, 0.56 +/- 0.16, 0.64 +/- 0.20 respectively. The mRNA and protein levels of RAR-beta had a significant difference between before and after interference with 5-Aza-cdR in SiHa, HeLa, and Caski cell lines (P < 0.05). However, they had no significant difference between before and after interference with 5-Aza-cdR in C33A cell line (P > 0.05). The 5-Aza-cdR treatment could suppress cell proliferation. CONCLUSIONS: The RAR-beta gene expression defects play an important role in the carcinogenesis of cervical cancer. Aberrant methylation in promotor region of RAR-beta gene may be an important mechanism for the loss of expression of RAR-beta gene.

[Expression of angiotensin II type 1 receptor in cervical squamous cell carcinoma and its clinical significance].

OBJECTIVE: To investigate the expression of Angiotensin II type 1 receptor (AT1R) in tissue and cell lines of squamous cervical carcinomas and its clinical significance, and to explore the molecular mechamisms of angiotensin II and AT1R activity in the process of cervical carcinogenesis. METHODS: (1) The levels of AT1R mRNA were examined by quantitative reverse transcriptase-polymerase chain reaction( RT-PCR) in paraffin-embedded tissues from 35 cases of cervical squamous cell carcinoma, 15 cases of cervical intraepithelial neoplasia (CIN), and 15 cases of normal cervix, and in Siha and C33a cells. The expression of AT1R protein in 65 specimens of cervix tissue sections was evaluated by immunohistochemistry. The corelation between the expressions of AT1R and its clinicopathologic features was analyzed accordingly. (2) After the Siha and C33a cells were treated at different concentrations of Angiotensin II (0, 10(-10) mol/L, 10(-9) mol/L, 10(-8) mol/L, 10(-7) mol/L, 10(-6) mol/L, 10(-5) mol/L) for different time in culture, the cell proliferation was determined by methylthiazolyl tetrazolium (MTT) assay. The vascular endothelial growth factor (VEGF) expression was examined by enzyme-linked immuno-absordent assay (ELISA). RESULTS: (1) AT1R mRNA expression was detected in the two cervix cancer cell lines. The positive rate of ATIR mRNA was 77.1%, 40.0% and 0, respectively, in squamous cell carcinomas, cervical intraepithelial neoplasia and normal cervical tissues, while their mRNA quantities were 0.3863 +/- 0.041, 0.0768 +/- 0.035 and 0, respectively. There was statistically a significant difference between them (P < 0.01). The average staining intensity of AT1R protein was stronger in invasive carcinoma cells than that in dysplasia tissues and normal ones (P < 0.01). Among 65 cases of squamous cell carcinomas, the expressions of AT1R mRNA and protein increased with pathological grading (P < 0.05), while it was neither correlated with clinical stage nor pelvic lymph node metastasis (P > 0.05). The level of AT1R protein expression corresponded to that of its mRNA. (2) Angiotensin II promoted the cell growth of cervical cancer cell lines Siha and C33a and induced secretion of VEGF from cells in a dose-dependent manner (P < 0.01), and the expression of VEGF was reversed by the addition of valsatan (an antagonist of angiotensin II type 1 receptor) (P < 0.01). CONCLUSION: Angiotensin II is involved in the progression of cervical carcinoma, since it may increase the proliferation activity of cancer cells, induce secretion of VEGF through AT1R synchronously, and results in an increase of angiogenesis in tumors. It suggests that use of AT1R antagonists may be an useful therapeutic strategy for cervical carcinoma.

[Reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel].

OBJECTIVE: To investigate the reversal effect of MDR1 and MDR3 gene silencing on resistance of A2780/taxol cells to paclitaxel. METHODS: shRNA plasmid vector specifically targeting MDR1 and MDR3 genes was transfected into A2780/taxol cells. The early stage cell apoptosis and the effect of intracellular rhodamine 123 (Rh123) accumulation were detected by flow cytometry (FCM). The late stage cell apoptosis rate was detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL). The 50% inhibition concentration (IC(50)) of paclitaxel on A2780/taxol cells was determined by methyl thiazolyl tetrazolium (MTT) assay. MDR1 and MDR3 mRNA were assessed by RT-PCR, and caspase-3 protein was detected by western blot. RESULTS: After treatment with MDR1 and MDR3 shRNA plasmid vector, early apoptosis rate of A2780/taxol cells was (20.21 +/- 0.56)% and (10.87 +/- 1.24)%, respectively. MDR1 and MDR3 shRNA could increase cellular Rh123 accumulation (116.6 +/- 8.1 and 98.4 +/- 3.8, respectively). The late stage apoptosis rates detected by TUNEL displayed the same tendency as FCM results did. The IC(50) for paclitaxel of A2780/taxol cells was decreased significantly. The mRNA levels of MDR1 and MDR3 in A2780/taxol cells were decreased by (73.3 +/- 0.8)% and (51.6 +/- 0.4)% of control, and the reduction of MDR1 and MDR3 mRNA was in a time-dependent manner. The expression of caspase-3 protein of MDR1 and MDR3 shRNA vector transfected group in A2780/taxol cells was significantly increased [(80.8 +/- 2.6)% and (72.0 +/- 4.7)%, respectively]. CONCLUSION: MDR1 and MDR3 gene silencing could recover sensitivity of A2780/taxol cells to paclitaxel and induce cell apoptosis, thus reversing cell resistance to paclitaxel.