Biological Characteristics and Molecular Mechanism of Procymidone Resistance in Stemphylium eturmiunum From Garlic.

Garlic leaf blight caused by Stemphylium eturmiunum was first reported in Jiangsu Province in China. The dicarboximide fungicide (DCF) procymidone is reported to possess broad-spectrum action in inhibiting filamentous fungi and is widely used to control leaf disease of various plants. Of 41 Stemphylium eturmiunum isolates collected in this study from commercial garlic farms in Pizhou and Dafeng counties of Jiangsu Province, eight isolates were resistant to procymidone. The following three phenotypes were categorized according to in vitro responses to DCFs: sensitive, low resistance to iprodione and procymidone, and high resistance to all iprodione and procymidone. The fitness of all resistant isolates was decreased in accordance with data on mycelial growth, conidiation, and virulence. After treatment with 10 µg/ml of procymidone for 4 h, mycelial intracellular glycerol concentrations of resistant isolates were significantly lower than those of sensitive isolates. Positive cross-resistance was observed between dicarboximides and phenylpyrroles, but there was no cross-resistance between dicarboximides and fluazinam or difenoconazole in the two resistant phenotypes. Nucleotide sequence alignment of two-component histidine kinase genes from sensitive and resistant isolates indicated that amino acid mutations were located at the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein and at the phosphatase domain of the N-terminal region and the response regulator domain of the C-terminal region. To our knowledge, this is the first report of DCF resistance in Stemphylium eturmiunum, and these findings will help establish a rational strategy to manage DCF-resistant populations of Stemphylium eturmiunum in the field.

Pazopanib Maintenance Therapy in East Asian Women With Advanced Epithelial Ovarian Cancer: Results From AGO-OVAR16 and an East Asian Study.

OBJECTIVE: The recent phase 3 trial AGO-OVAR16 demonstrated that pazopanib maintenance improved median progression-free survival in patients with ovarian cancer whose disease did not progress during first-line treatment. However, this improvement was not seen in the subset of East Asian patients. The current analysis evaluated the efficacy and safety of pazopanib maintenance in East Asian patients from AGO-OVAR16 and a separate East Asian study. MATERIALS AND METHODS: East Asian patients from AGO-OVAR16 (n = 209) and the East Asian study (N = 145) were randomized 1:1 to receive pazopanib 800 mg/d or placebo for up to 24 months. The primary end point for each study was progression-free survival by RECIST (Response Evaluation Criteria in Solid Tumors) based on investigator assessment. Clinical and genetics data were analyzed separately by study or pooled according to separate predetermined statistical plans. RESULTS: Pazopanib maintenance had a detrimental effect on median progression-free survival versus placebo in East Asian patients from the combined studies (n = 354; 17.9 vs 21.5 months; hazard ratio, 1.114; 95% confidence interval, 0.818-1.518; P = 0.4928). Pazopanib maintenance showed a disadvantage in overall survival in East Asian patients from AGO-OVAR16 versus placebo (hazard ratio, 1.706; 95% confidence interval, 1.010-2.883; P = 0.0465); overall survival analysis was not performed in the East Asian study because of insufficient event numbers. Pazopanib-treated patients had a significantly higher incidence of grade 3 or higher hypertension (27%) and neutropenia (13%) versus placebo. CONCLUSIONS: The treatment effect of maintenance pazopanib in East Asian patients seemed to differ from that in non-Asian patients. In study-specific and pooled analyses, none of the potential factors analyzed could satisfactorily explain the different efficacy results of pazopanib in East Asian patients.

Outcomes of fertility and pregnancy in patients with early-stage cervical cancer after undergoing neoadjuvant chemotherapy.

OBJECTIVE: To explore the outcomes of oncology, fertility, and pregnancy in patients after undergoing neoadjuvant chemotherapy (NACT) followed by fertility-sparing operations with cervical cancer, and its value in clinical treatment. MATERIALS AND METHODS: A total of 11 patients from seven hospitals in Beijing with cervical cancer since August 2009 to December 2011, who had undergone fertility- sparing treatments were recruited in this study. RESULTS: Among the 11 patients, there were nine cases of squamous cell carcinoma, two cases of adenocarcinoma, one case in Stage IA2, and ten cases in Stage IB1 (FIGO, 2009). All of the 11 patients were treated with NACT of one to two cycles before the operations, and then they underwent radical trachelectomy (RT) + retroperitoneal lymphadenectomy. Eleven patients had completed the follow-up (100%) and the mean follow-up was 24.4 months. The outcomes of the oncology and pregnancy are as follows: no patient recurred after fertility-sparing treatments; in seven patients seeking pregnancy after the treatments, three pregnancies occurred in two women. CONCLUSIONS: NACT+RT, as a fertility-sparing treatment for young women with bulky early-stage cervical cancer and its outcomes in fertility and pregnancy are satisfactory, however its safety needs to be studied further.

[Expression and significance of tumor necrosis factor receptor associated protein 1 in epithelial ovarian cancer].

OBJECTIVE: To explore the levels of TRAP1 and its roles in patients with ovarian tumor, and investigate the correlation between the expressions of TRAP1 in ovarian tumor tissues and related clinicopathological characteristics. METHODS: 38 health women, 50 cases of benign ovarian tumors and 114 cases of epithelial ovarian cancers were examined by real-time RT-PCR and immunohistochemical staining. RESULTS: The immunohistochemical analysis demonstrated that TRAP1 protein was mainly located in the cytoplasm, the protein and mRNA expression of TRAP1 in ovarian cancer were significantly increased compared with those of normal control and benign tumor (P < 0.05). The protein and mRNA expression of TRAP1 was related to histological grade and pathologic types (P < 0.05), but not age, clinical stages, lymphnode metastasis or omental metastasis, and the amount of ascites (P > 0.05). CONCLUSION: The high expression of TRAP1 may play potential role in epithelial ovarian cancer occurrence and progress.

Pien Tze Huang Inhibits Proliferation of Colorectal Cancer Cells through Suppressing PNO1 Expression and Activating p53/p21 Signaling Pathway.

OBJECTIVE: To explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it's down-stream mediators in colorectal cancer (CRC) cells. METHODS: Quantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues. RESULTS: PZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05). CONCLUSION: The mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21.

[Relationship between the expression of dual specificity phosphatase-1 and the prognosis of endometrioid adenocarcinoma].

OBJECTIVE: To clarify the relationship between the expression of dual specificity phosphatase-1 (DUSP1) and the prognosis of endometrioid adenocarcinoma. METHODS: The expression of DUSP1 was determined by immunohistochemical staining in specimens from 81 patients with endometrial carcinoma undergoing surgical resection. The relationship between DUSP1 expression, clinicopathological factors and prognosis were further evaluated. RESULTS: In 81 endometrioid carcinoma samples, 59 (72.84%) cases were positive while 22 negative.Except for lymph node metastasis, the expression of DUSP1 was correlated with FIGO stage, tumor grade, myometrial invasion and the expressions of estrogen receptor (ER) and progesterone receptor (PR) (P < 0.05) .Kaplan-Meier analysis showed that patients with a positive DUSP1 expression had significantly prolonged 5-year disease-free survival rates of 98.2% and 76.0% respectively (P < 0.05). And COX regression analysis revealed that the expressions of DUSP1 and PR were independent prognostic indicators of endometrioid carcinoma, the HR (hazard ratio) of DUSP1 negative expression was 21.2.Spearman analysis further showed that the expression of DUSP1 was positively correlated with PR (r = 0.256, P < 0.05). CONCLUSION: DUSP1 may be a potential negative prognostic indicator for endometrioid adenocarcinoma.

[Study of mechanism of medroxyprogesterone 17-acetate on the cancer stem cell-like properties of human endometrial cancer].

OBJECTIVE: To explore the mechanism resistance of medroxyprogesterone 17-acetate(MPA) on the endometrial cancer side-population(SP) cells. METHODS: (1) Ishikawa-SP cells from endometrial cancer cell lines Ishikawa were be separated by Hoechst 33342 dyeing method and flow cytometry analysis. The clone formation efficiency between Ishikawa-SP cells and Ishikawa-non-SP cells were performed by clone formation assay. Breast cancer resistance protein (BCRP) was examined by immunocytochemistry method. (2) Ishikawa, Ishikawa-SP, Ishikawa-non-SP cells were treated with various concentrations of MPA at 5, 10, 15, 20 µmol/L. After cultured for 24, 48, and 72 hours, cells growth were measured by methanethiosulfomate (MTS) assay. (3) The groups of Ishikawa, Ishikawa-SP, Ishikawa-non-SP cells incubated with MPA at the half maximal inhibitory concentration (IC50) were selected for cell apoptosis assay by using flow cytometry. After MPA treatment, the expression of caspase-3 was examined by immunocytochemistry method. RESULTS: (1) There were few proportion of Ishikawa-SP cells in Ishikawa endometrial carcinoma, which were 2.7%. There were stronger clone formation efficiency for Ishikawa-SP cells than that for Ishikawa-non-SP cells in Ishikawa [(6.02 ± 1.17)% vs.(0.53 ± 0.20)%, P = 0.001]. And there were higher level expression of BCRP (P = 0.001) and also more resistant Taxol and radiation between Ishikawa-SP cells and Ishikawa-non-SP cells. (2) The inhibitory effect of MPA was concentration-dependent and time-dependent. (3)After MPA treatment, the apoptosis rates of Ishikawa-SP, Ishikawa-non-SP,Ishikawa were (4.01 ± 0.43) %, (9.30 ± 0.67) %, and (4.64 ± 0.18) %, respectively(P < 0.05). The level expression of caspase-3 in Ishikawa group after MPA treated were higher than that in Ishikawa-SP group. CONCLUSION: MPA may be inhibit the growth of endometrial cancer, Ishikawa-SP and Ishikawa-non-SP cells, while Ishikawa-SP may be more resistant to MPA than Ishikawa-non-SP, which mechanism of resistance on MPA may be related to the properties of cancer stem-like cells and cell apoptosis.

[Application of endometrial sampling device during the follow-up visit for the conservative treatment of endometrial cancer].

OBJECTIVE: To study the feasibility of endometrial sampling device as a sampling tool during the follow-up visit for endometrial cancer patients undergone conservative treatment. METHODS: Before the hysteroscopy examination, endometrial sampling device was used to take the endometrium specimens 43 times in 19 patients who had been diagnosed as endometrial cancer or atypical hyperplasia, and were undergone conservative treatment during May 2012 to Mar. 2013. All cases accepted vaginal ultrasound screening before every sampling by endometrial sampling device. The histological results were compared with those done by hysteroscopy. RESULTS: The average age of those patients was (30 ± 6) years old. The mean thickness of the endometrium during the treatment was (0.81 ± 0.65) cm. The qualified rate for the sampling was 95% (41/43). Compared with the specimens undergone by hysteroscopy direct sampling, 32 samples got by the endometrial sampling device with thicker endometrium (0.93 ± 0.70) cm had the same histological results, while the other 9 patients with thinner endometrium (0.40 ± 0.14) cm were not (P = 0.031). CONCLUSION: The endometrial sampling device could be used during the follow-up visit for the conservative treatment patients with endometrial cancer or atypical hyperplasia, the vaginal ultrasound screening should be used together to figure out those with thinner endometrium.

Dynamic alteration and prognostic significance of tumor-associated CD68+ and CD68+ PD-L1- macrophages in muscle-invasive bladder cancer treated with neoadjuvant chemotherapy.

BACKGROUND: The current study aimed to investigate the dynamic alteration and prognostic significance of tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and PD-L1 status of immune cells in muscle-invasive bladder cancer (MIBC) treated with neoadjuvant chemotherapy (NAC). METHODS: Multiplex immunofluorescence staining was performed to examine CD68+ TAM, CD4+ T cell, CD8+ T cell, FOXP3+ Treg cell, and PD-L1 expression in paired MIBC tissues (n = 54) before and after NAC. Patients were then divided into definite responders (DR), (≤pT1) and incomplete responders (IR). RESULTS: There was no significant difference between DR and IR cohorts for the immune cell infiltration levels at the baseline status. Tobacco history was identified to be associated with worse NAC efficacy. CD68+ (stroma area: p = 0.025; tumor area: p = 0.028; total area: p = 0.013) and CD68+ PD-L1- (stroma area: p = 0.035; tumor area: p = 0.013 total area: p = 0.014) TAMs infiltration levels decreased significantly after NAC, while there was no significant difference of CD68+ PD-L1+ and TILs. The infiltration of CD68+ (p = 0.033), CD68+ PD-L1- (p = 0.033), and CD68+ PD-L1+ (p < 0.001) TAMs in stroma area were significantly associated with poorer disease-free survival rate (DFS) of MIBC patients. CONCLUSION: CD68+ and CD68+ PD-L1- TAMs infiltration levels decreased significantly after NAC and pre-treatment TAM infiltration levels were independent prognostic factors for MIBC patients. While there was no sufficient evidence demonstrating that pre-treatment TILs or TAMs could predict response to NAC in MIBC patients.

Implementation of visual inspection with acetic acid and Lugol's iodine for cervical cancer screening in rural China.

OBJECTIVE: To address the value of visual inspection where HPV-based screening is not yet available, we evaluated the real-world effectiveness of visual inspection with acetic acid (VIA) and with Lugol's iodine (VILI) as a primary screening method for cervical cancer in rural China. METHODS: A total of 206 133 women aged 30-59 years received two rounds of VIA/VILI screening for cervical cancer in 2006-2010. Women with positive screening results underwent colposcopy and direct biopsy, and were treated if cervical intraepithelial neoplasia grade 2 or worse (CIN2+) was diagnosed. Clinical effectiveness of VIA/VILI was evaluated by process and outcome measures. RESULTS: The VIA/VILI positivity rate, biopsy rate and detection rate of CIN2+ in the second round were significantly lower than in the first round. The 2-year cumulative detection rate of CIN2+ varied from 0.53% to 0.90% among the four cohorts initiated in 2006, 2007, 2008, and 2009. The first round of screening detected 60%-83% of CIN2, 70%-86% of CIN3, and 88%-100% of cervical cancer. Over 92% of CIN2+ were found at the early stage. CONCLUSION: Multiple rounds of visual inspection with continuous training and quality assurance could act as a temporary substitutional screening method for cervical cancer in resource-restricted settings.

Prevalence of human papillomavirus and cervical intraepithelial neoplasia in China: a pooled analysis of 17 population-based studies.

High-risk (HR) human papillomavirus (HPV) prevalence has been shown to correlate well with cervical cancer incidence rates. Our study aimed to estimate the prevalence of HR-HPV and cervical intraepithelial neoplasia (CIN) in China and indirectly informs on the cervical cancer burden in the country. A total of 30,207 women from 17 population-based studies throughout China were included. All women received HPV DNA testing (HC2, Qiagen, Gaithersburg, MD), visual inspection with acetic acid and liquid-based cytology. Women positive for any test received colposcopy-directed or four-quadrant biopsies. A total of 29,579 women had HR-HPV testing results, of whom 28,761 had biopsy confirmed (9,019, 31.4%) or assumed (19,742, 68.6%) final diagnosis. Overall crude HR-HPV prevalence was 17.7%. HR-HPV prevalence was similar in rural and urban areas but showed dips in different age groups: at age 25-29 (11.3%) in rural and at age 35-39 (11.3%) in urban women. In rural and urban women, age-standardized CIN2 prevalence was 1.5% [95% confidence interval (CI): 1.4-1.6%] and 0.7% (95% CI: 0.7-0.8%) and CIN3+ prevalence was 1.2% (95% CI: 1.2-1.3%) and 0.6% (95% CI: 0.5-0.7%), respectively. Prevalence of CIN3+ as a percentage of either all women or HR-HPV-positive women steadily increased with age, peaking in 45- to 49-year-old women. High prevalence of HR-HPV and CIN3+ was detected in both rural and urban China. The steady rise of CIN3+ up to the age group of 45-49 is attributable to lack of lesion removal through screening. Our findings document the inadequacy of current screening in China while indirectly raising the possibility that the cervical cancer burden in China is underreported.

Hypomethylated CpG around the transcription start site enables TERT expression and HPV16 E6 regulates TERT methylation in cervical cancer cells.

OBJECTIVE: The human papillomavirus (HPV) oncoprotein, E6, activates telomerase reverse transcriptase (TERT) expression and causes cellular immortalization. It remains unclear whether E6 affects TERT transcription by altering DNA methylation profiles. In this study, we explored the methylation status of the TERT promoter in cervical cancer cell lines and its variations after E6 was silenced by RNAi. METHODS: Three kinds of cervical cell lines (HPV16 positive: CaSki and SiHa; HPV18 positive: HeLa), were taken to analyze the methylation status of the TERT promoter by methylation-specific polymerase chain reaction (MSP) and bisulfite sequencing (BS). Stealth RNAi was transiently transfected to these cell lines to silence the expression of HPV16/18 E6, and the subsequent changes of TERT mRNA levels and TERT promoter DNA methylation were examined. RESULTS: Hypomethylation of the DNA around the TERT transcription start site (-156 to +162 bp) was functionally related to its transcription. After transfection with Stealth RNAi, the levels of HPV16/18 E6 and TERT mRNA were greatly decreased. The methylated CpG around the transcription start sites in CaSki and SiHa cells were statistically increased (respectively P=0.016, P=0.000). However, there was no significant difference in HeLa cells (P=0.128). CONCLUSION: Hypomethylated CpG around the transcription start site enables the expression of TERT in cervical cancer cells. Our results show for the first time that HPV16 E6 can promote TERT transcription through demethylating the DNA sequence around the TERT transcription start site in cervical squamous cancer cells.

Value-based medicine analysis on loop electrosurgical excision procedure and CO2 laser vaporization for the treatment of cervical intraepithelial neoplasia 2.

AIM: The best treatment option for cervical intraepithelial neoplasia 2 (CIN2) is controversial and there is a lack of studies in value-based medicine. This multicenter comparative study was undertaken to evaluate the effectiveness, cost-effectives and quality of life (QOL) of loop electrosurgical excision procedure (LEEP) and CO(2) laser vaporization for the treatment of CIN2. MATERIAL AND METHODS: A database of LEEP and laser vaporizations performed at three research centers was created. Patients with colposcopic-histopathologically confirmed CIN2 were randomly submitted to LEEP and laser vaporization. Cytology, human papilloma virus (HPV) DNA test and histology were performed, and a questionnaire on QOL was filled out during follow-up. Effectiveness, cost-effectives and QOL were analyzed. RESULTS: Three hundred and thirty-eight women with CIN2 were included in the study. Frequencies of remission, and persistent and recurrent CIN were 89.2%, 7.2%, and 3.6% for LEEP, and 86.7%, 12.6%, 0.70% for laser, respectively. There was no significant difference in remission and persistence of CIN. There was a significant difference in the number of operations, recovery time and costs. Women treated with two methods showed relatively identical QOL. CONCLUSION: Both LEEP and CO(2) laser vaporization are effective and reliable treatments for CIN2, whereas cervical tissue can be obtained for histology by LEEP. Preoperative evaluation and postoperative follow-up are important. Gynecologists should pay attention to QOL of patients with CIN.

[Changes in awareness of cervical cancer patients after medical treatments and the correlation of cervical screening situation and clinical stage at consultation].

OBJECTIVE: To Investigate the cervical cancer patients' knowledge and situation concerning cervical cancer screening and explore the connection of cervical cancer screening status and clinical stage at diagnosis of cervical lesions. METHODS: Questionnaires were done for the cervical cancer patients who went to Peking University People's Hospital from February 2010 to October 2011. The patient's age, first visit or not, cause for treatment, awareness of cervical cancer now as well as that before diagnosis of the disease, the last cervical cancer screening' time and the clinical stage were recorded. The results of questionnaires were analyzed with SPSS 16.0. RESULTS: (1) One hundred and thirty-eight cases were collected and the average age was (48 ± 10) years old. Concerning the awareness level, the patients were divided into three groups of 10 points, 5 points and 0 point based on the score of the second parts of the questionnaires. The number of the patients of the above three groups were 114 (82.6%), 18 (13.0%) and 6 (4.3%) at present, while before the cancer diagnosis the number of the patients of the above three groups were 5 (3.6%), 13 (9.4%) and 120 (87.0%). Patient's awareness level was much higher now than that before the cancer diagnosis (P < 0.05). (2) Based on the last cervical cancer screening time, the patients were divided into four groups including within one year (group 1), a year ago but within three years (group 2), three years ago (group 3) and never done (group 4). There were 9 (6.5%) cases in group 1, 30 (21.7%) cases in group 2, 29 (21.0%) cases in group 3 and 70 (50.7%) cases in group 4. There were 5 (5/9) cases with stage Ia, 3 (3/9) cases with stage Ib and 1 (1/9) case with stage II or above in group 1; 11 (36.7%, 11/30) cases with stage Ia, 15 (50.0%, 15/30) cases with stage Ib and 4 (13.3%, 4/30) cases with stage II or above in group 2; 5 (17.2%, 5/29) cases with stage Ia, 14 (48.3%, 14/29) cases with stage Ib was and 10 (34.5%, 10/29) cases with stage II or above in group 3; 7 (10.0%, 7/70) cases stage with Ia, 35 (50.0%, 35/70) cases with stage Ib and 28 (40.0%, 28/70) cases with stage II or above in group 4. The results showed that there was an increasing trend of advanced cervical carcinoma at diagnosis for the patients with longer last cervical cancer screening time (P < 0.05). CONCLUSIONS: People's awareness level is much higher after being educated in cervical cancer. Among the cervical cancer patients, the probability of diagnosed patients with advanced cervical cancer has an increasing trend when their screening situation is poor. To raise the screening attendance rate among appropriate women, we should spread knowledge of cervical cancer to the population.

[Impact of calcium channel antagonists for estrogen action on the endometrial carcinoma HEC-1A cells].

OBJECTIVE: To study the effects of nifedipine and mibefradil on the cell proliferation, apoptosis, migration on the HEC-1A in vitro and also study the mRNA and protein expression levels of the calcium channel alpha1D (Cav1.3) and calcium channel alpha1G (Cav3.1) to discuss the effects of the calcium antagonists on the mechanisms of the endometrial carcinoma. METHODS: (1) Add 10 µmol/L nifedipine and mibefradil at 15 minutes before adding 10 µmol/L 17ß-estradiol (17ß-E(2)) and 100 µmol/L b-estradiol-6-(O-carboxymethyl)oxime (E(2)-BSA) to the HEC-1A in different time including 0, 5, 15, 30, 60, 120 minutes. Then the changes of mRNA and protein were detected by reverse transcripiton (RT)-PCR and western-blot. (2) Add 1.25, 2.5, 5, 10, 20, 40, 80, 100 µmol/L nifedipine and mibefradil to the HEC-1A at 24, 48, 96 hours to detect the cell proliferation by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method. (3) Add 10 µmol/L nifedipine and mibefradil to the HEC-1A, then detect the apoptosis at 0 minute, 30 minutes, 1 hour, 6 hours, 24 hours and migration in vitro at 36 hours with transwell methods. RESULTS: (1) After the pretreated effect of the nifedipine before 17ß-E(2), the mRNA express of Cav1.3 genes was lowest at 15 minutes, and returned to the control level after 30 minutes. The protein level didn't change very much in 30 minutes, but rose after 60 minutes. The Cav3.1 genes mRNA express was lowest at 5 minutes, rose at 30 minutes and returned to the 0 minute level gradually. (2) After the pretreated effect of the nifedipine before E(2)-BSA, the Cav1.3 genes mRNA was lowest at 5 minutes and returned at 15 minutes. The protein level rose gradually in 15 minutes but reduced after 15 minutes. The Cav3.1 mRNA and protein level were reduced at every time point. (3) After the pretreated effect of the mibefradil before 17ß-E(2), there was no change of mRNA expression of Cav1.3 genes. The protein level rose at 15 and 60 minutes, there was no change in any other time. The Cav3.1 genes mRNA were gradually reduced and the protein level rose at 15 minutes, and there was no change in any other times. (4) After the pretreated effect of the mibefradil before E(2)-BSA, the mRNA and protein of Cav1.3 levels were reduced after 15 minutes. There was no mRNA expression of Cav3.1, while the protein level was lowest at 15 minutes. (5) Nifedipine and mibefradil affected HEC-1A proliferation depended on the different concentration and interval time points. There was significant difference than those in control group (P < 0.05). (6) There were statistical differences in apoptosis rate after adding nifedipine (P < 0.05), while rose at mibefradil treated the same time (24 hours: 8.41 ± 0.07, 0 minute: 3.74 ± 0.18; P < 0.05). (7) The numbers of stained cells after both nifedipine and mibefradil treated reduced more than control group. CONCLUSIONS: (1) Nifedipine and mibefradil could inhibit both the effect of the estrogen on the L-type and T-type calcium channel in short time, meanwhile the mibefradil effects last long time. (2) The inhibited effect of the mibefradil on the proliferation, apoptosis and migration of HEC-1A cells in vitro is more significant than that by nifedidipine.

[Preliminary investigation of the expression and functions of insulin receptor isoforms in endometrial carcinoma].

OBJECTIVE: To investigate the expression of insulin receptor isoforms (IR, including IR-A and IR-B) in endometrial carcinoma (EC), and explore the role of IR-A in the growth of endometrial carcinoma cells. METHODS: The expression of IR isoforms were detected by reverse transcription (RT)-PCR and real-time PCR in 4 different endometrial cancer cell lines (HEC-1-A, Ishikawa, RL95-2 and KLE), with human breast cancer cell line MCF-7 cells and hepatocellular carcinoma cell line Hep-G2 as positive control and in the endometrial cancer tissue specimens of 42 cases, admitted in Peking University People's Hospital from November 2007 to July 2009, with normal endometrial tissues from 15 cases of ovarian neoplasms at the same period as controls. The relationships among the expression of IR, IR-A isoforms and the clinicopathological parameters of EC tissues were analyzed by Spearman rank correlation analysis. An eukaryotic IR-A expression plasmid was constructed and transfected into RL95-2 cells [RL95-2(IR-A)] for overexpression of IR-A in RL95-2 cells, in which the expression of IR-A originally was low. Non radioactive cell proliferation assay-MTS was used to determine the proliferation curves in the four EC cells listed above and in RL95-2 or RL95-2 (IR-A). RESULTS: (1) There were two isoforms of IR-A and IR-B co-expressed were detected in EC cells and EC tissues. Among four kinds of EC cell lines, the expression level of IR mRNA in RL95-2 cells was the highest, followed by Ishikawa, KLE and HEC-1-A cells, in which the relative IR mRNA expression levels were (26.54 ± 1.82)×10(-4), (15.44 ± 3.29)×10(-4), (10.14 ± 0.10)×10(-4) and (2.63 ± 0.23)×10(-4), respectively (P < 0.01). The expression level of IR-A mRNA was the highest in Ishikawa cells, followed by KLE, RL95-2 and HEC-1-A cells, with the relative expression levels were (12.07 ± 3.31)×10(-4), (4.68 ± 0.63)×10(-4), (3.03 ± 0.22)×10(-4) and(1.46 ± 0.03)×10(-4), respectively (P < 0.01). The relative expression level of IR and IR-A mRNA were 0.017 ± 0.013 and 0.011 ± 0.010 in the EC tissues, respectively, compared with 0.015 ± 0.014 and 0.010 ± 0.012 in the controls, in which there were no significant differences in the expression level of IR or IR-A mRNA between EC tissues and the control (P = 0.662, P = 0.780). The expression of IR and IR-A in EC tissues had no significant relevance with International Federation of Gynecology and Obstetrics (FIGO) stage, cell differentiation, depth of myometrial invasion, invasion of lymph-vascular space, lymph nodes metastasis, the expression status of estrogen receptor, human progesterone receptor, and PTEN gene (all P > 0.05). The expression of IR-A mRNA in EC patients with type 2 diabetes mellitus (DM) was significantly higher than that in patients without type 2 DM (P = 0.031), while there were no statistical correlation between the expression of IR mRNA and type 2 DM (P = 0.438). (2) The proliferation rates of the four kinds of EC cells was positively related with the IR-A expression ratio, with the most growth potential in Ishikawa, followed by HEC-1-A, KLE and RL95-2 cells. The overexpression of IR-A in RL95-2 (IR-A) cells showed a significant proliferation-promoting effect than that in control RL95-2 cells (P < 0.01). CONCLUSION: There are two isoforms of IR-A and IR-B co-expressed in EC. The overexpression of IR-A may promote the proliferation of EC cells.

[Value of CA(125) in the prediction of optimal interval debulking surgery and its prognosis in patients with epithelial ovarian cancer].

OBJECTIVE: To investigate the changes of CA(125) between primary cytoreductive surgery and interval debulking surgery for prediction the rate of optimal interval cytoreductive surgery and prediction the recurrence and the prognosis in patients with epithelial ovarian cancer. METHODS: A total of 39 cases with suboptimal primary cytoreductive surgery admitted from Jan. 1996 to Jan. 2009 were retrospectively analyzed. The median age of patients was 56 years (range: 41 - 68 years). Based on the changes in CA(125) level between primary cytoreductive surgery and interval debulking surgery, all cases were divided into four groups, group A (CA(125) reduced to normal after primary cytoreductive surgery, n = 6), group B (CA(125) reduced to normal after 1-2 cycles of chemotherapy, n = 11), group C (CA(125) reduced to normal after 3-4 cycles of chemotherapy, n = 14), and group D (CA(125) did not reduced to normal after the chemotherapy, n = 8), and all received platinum-based chemotherapy. The response to chemotherapy evaluated by pathological examination versus CA(125) level, and recurrence and prognoses were also analyzed. RESULTS: (1) The rate of optimal interval cytoreductive surgery in group A, B, C and D were 6/6, 8/11, 9/14 and 2/8 respectively, in which there were statistically different between group A or B and group D (P < 0.05). (2) The clinical benefit rates evaluated by the pathological examination in group A, B, C and D were 4/6, 4/11, 5/14 and 0, respectively and there were statistically different between group A and group D (P = 0.030). (3) There was significant difference in the recurrence rate between group A and group D (3/6 vs. 8/8, P = 0.024), while there were not significant differences between group B or C and group D (all P > 0.05). The rate of drug-resistant recurrence in group A, B, C and D were 1/6, 3/11, 5/14 and 7/8, respectively, in which there were significant differences between group A, B or C and group D (all P < 0.05). (4) The median progression-free survival (PFS) for patients in group A, B, C and D were 32, 10, 18 and 3 months, respectively, in which there were significant differences in the PFS between group A, B or C and group D (P = 0.012, P = 0.003, P = 0.032). The median overall survival (OS) were 44, 45, 44 and 16 months, respectively. There were significant differences in the OS between group A, B or C and group D (P = 0.022, P = 0.004, P = 0.000). CONCLUSION: The change of CA(125) between primary cytoreductive surgery and interval debulking surgery may be predict the recurrence type and the prognosis in patients with epithelial ovarian cancer.

[Re-evaluation of interval debulking surgery in advanced epithelial ovarian cancer].

OBJECTIVE: Previous study showed that interval debulking surgery (IDS) may improve the survival of patients with advanced epithelial ovarian cancer (EOC). The precise significance of IDS needs to be evaluated. METHODS: Totally 136 consecutive patients with stage IIIc or IV EOC (including primary peritoneal carcinoma and primary fallopian tube carcinoma) who completed primary debulking surgery (PDS) and platinum-based chemotherapy were enrolled from January 2000 to December 2009 in a retrospective cohort study. The study group was divided into three groups: 65 cases underwent optimal PDS (Group A), 41 cases received chemotherapy alone after suboptimal PDS (Group B), and 30 patients underwent IDS after suboptimal PDS (Group C). All patients received six to eight courses of platinum-based combination chemotherapy (paclitaxel plus carboplatin/cisplatin, cyclophosphamide plus epirubicin and cisplatin). Patients' clinical characteristics, perioperative situation and prognosis were compared. RESULTS: Sixty-five cases (47.8%, 65/136) from 136 patients achieved optimal PDS. For Group C, 77% (23/30) patients obtained optimal debulking surgery after IDS. Intraoperative injury rates were similar between Group B and Group C (P > 0.05). Mild perioperative complications rate was also similar (P > 0.05). Median progression-free survival (PFS) of Group A was 26 months. Median overall survival (OS) of Group B and Group C were 31 months and 40 months, respectively (P = 0.254). Median PFS of Group B and Group C were 13 months and 24 months, respectively (P = 0.289). Although when it came to 20 months after PDS, patients who underwent IDS had a significantly lower progressive disease (PD) rate (Group B 33% versus Group C 61%, P = 0.046), it still showed that there was no significant difference in either OS or PFS of these two groups. Those patients in Group C who obtained no visible residual got similar PFS (27 months) comparing to Group A (26 months, P = 0.730), but OS was still shorter (P = 0.010). CONCLUSIONS: For advanced EOC patients, IDS has little effect on improving survival. While it is safe and acceptable, also may prolong PFS in those patients who got no visible residual after IDS. The results suggest that IDS might be used as an alternative treatment for advanced EOC patients who cannot obtain optimal PDS in certain local hospitals.

[Significance of prognostic evaluation of International Federation of Gynecology and Obstetrics 2009 staging system on stage I endometrioid adenocarcinoma].

OBJECTIVE: To explore the impact of 2009 International Federation of Gynecology and Obstetrics (FIGO) staging system alteration for stage I endometrioid adenocarcinoma on its' prognosis assessing. METHODS: A retrospective study was carried out on 244 cases with endometrial carcinoma admitted in Peking University People's Hospital from Jan.1995 to Feb.2008. RESULTS: (1) All 244 patients were divided into FIGO 2009 Ia group (n = 200) and FIGO 2009 Ib group (n = 44) according to FIGO 2009 staging system, while they were divided into FIGO 1988 Ia group (n = 34), FIGO 1988 Ib group (n = 156) and FIGO 1988 Ic group (n = 29). The others 25 cases were stage IIa (n = 16) and stage IIIa with merely positive abdominal cytology (n = 9) according to FIGO 1988 staging system.(2) The higher percentage of low-grade in FIGO 1988 Ia group than that in FIGO 2009 Ia group (P = 0.003). Compared with FIGO 2009 Ia group, the age of the patients, surgery extent, the percentage of lymph node excision and received chemotherapy and radiotherapy, there were no difference in FIGO 1988 Ia and Ib group, respectively (P > 0.05). There were 5.9% (2/34) and 6.7% (10/150) found relapse among FIGO 1988 Ia group and FIGO 1988 Ib group, and there were 2.9% (1/34) and 2.7% (4/150) for the two groups died of carcinoma. Compared with FIGO 2009 Ia group, there were not significant difference [7.5% (13/200) vs. 3.0% (6/200); P > 0.05]. The 5 years and 10 years progression-free survival (PFS) of FIGO 1988 Ia group and Ib group were (97.0 ± 3.0)%, (90.9 ± 6.5)% and (95.3 ± 2.1)%, (90.2 ± 3.6)%, respectively, in which there were not significant difference compared with that in FIGO 2009 Ia group [(96.1 ± 1.6)%, (89.6 ± 3.2)%; P > 0.05]. The 5 years and 10 years overall survival (OS) in FIGO 1988 Ia group and Ib group were 100%, (93.8 ± 6.0)% and (96.9 ± 1.8)%, (95.2 ± 2.5)%, respectively, in which there were did not significant difference with that in FIGO 2009 Ia group [(97.9 ± 1.2)%, (93.4 ± 2.8)%; P > 0.05].(3) There were not significant difference between FIGO 1988 Ic group and FIGO 2009 Ib group (P > 0.05) for the age of the patients, grade, surgery extent, lymph node excision, the percentage of received chemotherapy and radiotherapy. Between FIGO 1988 Ic group and FIGO 2009 Ib group, there were 3.4% (1/29) and 6.8% (3/44) cases found relapse, respectively. And there were 0 and 2.3% (1/44) cases died of carcinoma in the two groups, in which there were not differ much either (P > 0.05). The 5 years and 10 years PFS in FIGO 1988 Ic group were all 100%, while they were 100% and (90.9 ± 6.2)% in FIGO 2009 Ib group. The 5 years and 10 years OS in FIGO 1988 Ic group were all 100%, but were 100% and (95.0 ± 4.9)% in FIGO 2009 Ib group, in which they all did not significantly differ much (P > 0.05). (4) The patients in FIGO 2009 Ia group were younger than those in FIGO 2009 Ib group (P < 0.01). The percentage of low grade in FIGO 2009 Ia group were higher than that in FIGO 2009 Ib group (P = 0.029). The percentages of received chemotherapy and radiotherapy in FIGO 2009 Ia group were lower than that in FIGO 2009 Ib group remarkably (P < 0.01). But there were not significant difference in the uterine excision extent and the percentage of lymph node excision between the two groups (P > 0.05). There were not significantly differ in the relapse rates and the death rates between the FIGO 2009 Ia group and FIGO 2009 Ib group (P > 0.05). There were also not significant difference in PFS and OS between the two groups (P > 0.05). CONCLUSIONS: There were not significant difference in the prognosis between FIGO 2009 stage Ia and FIGO 1988 stage Ia and Ib. There were also not significant difference in the prognosis between FIGO 2009 stage Ia and FIGO 2009 stage Ib, which may be due to received more chemotherapy and radiotherapy in FIGO 2009 stage Ib patients.

Circular RNA circ_0023404 serves as a miR-636 sponge to promote malignant behaviors in cervical cancer cells through upregulation of CYP2S1.

Cervical cancer is the most common malignant gynecological tumor. Circular RNA (circRNA) circ_0023404 is reported to be upregulated in cervical cancer cells. This aim is to explore the role and mechanism of circ_0023404 in cervical cancer. circ_0023404, microRNA-636 (miR-636), and cytochrome P450 2S1 (CYP2S1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell proliferation, migration, invasion, and apoptosis were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, 5-ethynyl-2'-deoxyuridine (EDU) assay, colony formation assay, transwell assay, and cytometry assay. Protein levels of cyclin D1, matrix metallopeptidase 9 (MMP9), Bcl-2-associated X protein (Bax), and CYP2S1 were examined by western blot assay. The binding relationship between miR-636 and circ_0023404 or CYP2S1 was predicted by Circinteractome or targetscan, and then verified by a dual-luciferase reporter assay and RNA pull-down assay. circ_0023404 and CYP2S1 expression were increased, and miR-636 was decreased in cervical cancer tissues and cells. Moreover, circ_0023404 knockdown could repress proliferation, migration, invasion, and promote apoptosis of cervical cancer cells in vitro. Mechanically, circ_0023404 could regulate CYP2S1 expression by sponging miR-636. circ_0023404 silencing could attenuate the progression of cervical cancer cells partly by targeting the miR-636/CYP2S1 axis, hinting at a promising therapeutic target for cervical cancer.