Safety and Efficacy of Neoadjuvant Chemotherapy Followed by Radical Surgery Versus Radical Surgery Alone in Locally Advanced Cervical Cancer Patients.

OBJECTIVES: This study aimed to evaluate the safety and efficacy of neoadjuvant chemotherapy (NACT) followed by radical surgery (RS) among patients with locally advanced cervical cancer (LACC). METHODS: Eight hundred patients with LACC received either NACT followed by RS (NACT-RS) or RS alone. The primary outcome measures assessed the efficacy and adverse effects of NACT. Secondary outcome measures compared the preoperative clinical stage to the postoperative pathologic stage in NACT-RS and RS patients, assessed intraoperative and postoperative complications, including the adverse effects of postoperative radiotherapy and radiochemotherapy, and estimated the 5-year progression-free survival and 5-year overall survival. RESULTS: The clinical response to NACT was 89.54%. Patients in the NACT-RS group had lower preoperative hemoglobin levels (115.20 vs 122.04 g/L, P < 0.001), a longer operative time (mean, 233.66 vs 224.37 minutes, P = 0.008), more intraoperative bleeding (750.34 vs 684.41 mL, P = 0.011), a shorter duration of catheter use (mean, 29.84 vs 32.14 days, P = 0.036), and a lower incidence of postoperative complications (7.30% vs 13.62%, P = 0.002) and postoperative radiotherapeutic and radiochemotherapeutic adverse effects (3.16% vs 4.63%, P < 0.001) compared to patients in the RS group. The 5-year progression-free survival and 5-year overall survival were 80.30% and 81.10% in the NACT-RS group and 81.00% and 78.50% in the RS group (P > 0.05). Pathological poor differentiation, nonsquamous cell carcinoma, parametrial invasion, positive pelvic lymph node, and lymphovascular invasion (P < 0.05) were independent risk factors for recurrence. CONCLUSIONS: Neoadjuvant chemotherapy may reduce RS-associated complications and postoperative radiotherapeutic and radiochemotherapeutic adverse effects in Chinese patients with LACC.

[HPV E7 Function in DNA Damage Response of Cervical Intraepithelial Neoplasia.]

OBJECTIVES: To determine the function of human papillomavirus (HPV) E7 in DNA damage response of cervical intraepithelial neoplasia (CIN) 3 cells. METHODS: Samples of CIN 3 and child foreskin tissues were collected,with pathologically confirmed HPV positive and negative,respectively.Collagenase A was used for digesting tissues prior to primary culture.The HPV negative cells were infected with lentivirus E7 and pLV.Proteins(53BP1,NBS1,BRCA1 and RPA32) responsive to DNA double break damages were detected by indirect immunofluorescent staining after 0-8 h treatment with X-ray (2 or 5 Gy). RESULTS: After treatment with 2 or 5 Gy X-ray,53BP1,NBS1,BRCA1 and RPA32 foci in HPV+ cells increased compared with HPV- cells (P<0.05).Less 53BP1,RPA32,BRCA1 and NBS1 foci positive cells (foci>5) were found in E7 infected cells than in pLV infected cells(P<0.05).Both of them reached the peak at 6 h (2 Gy) or 4 h (5 Gy). CONCLUSIONS: We have successfully established a model to detect the function of HPV E7 in DNA damage response using primary culture of CIN fibroblasts.With the progression of CIN,HPV E7 can inhibit DNA double break repair.

[Employing DNA Damage Response Inhibitors to Enhance Chemosensitivity of Ovarian Carcinoma Cells].

OBJECTIVE: To assess the sensitisation effects of DDR inhibitors combined with conventional chemotherapeutics agents (cisplatin et al) in a drug-resistant ovarian cancer cell line(OVCAR-8). METHODS: Inhibitors of DDR regulators with cisplatin were applied to challenge OVCAR-8, and evaluated the DNA damage response (DDR) and cytotoxic effects of different combination of chemicals. Inhibition of proliferation to OVCAR-8 of different drugs was evaluated by MTT assay. The activation of phosphorylation of histone family 2A variant (yH2AX) and p53 binding protein 1 (53BP1) in OVCAR-8 were evaluated by immunofluorescence to observe their ability of recruitment and forming foci at DNA damage site. RESULTS: We observed that combined treatment of ataxia-telangiectasia mutated (ATM)/ATM and Rad 3-related (ATR) inhibitor and cisplatin can suppress the activation of damage repair mechanisms and weakened the proliferative activity of OVCAR-8 cells (P<0. 01) ; ATR pathway was suppressed and the signal of γH2AX weakened and cell survival rate significantly reduced when combination therapy of HU and Wortmannin (P < 0.05); poly ADP-ribose polymerase (PARP) inhibitor could not enhance chemosensitivity in OVCAR-8 cells when combined with cisplatin (P > 0.05). CONCLUSION: We substantiated that appropriate inhibitors of DNA damage response may have a potential to improve the anti-tumor effect of conventional chemotherapy drugs and prevent drug resistances.

[Functional Analysis of DNA Damage Repair Factor WDR70 and Its Mutation in Ovarian Cancer].

OBJECTIVES: To analyze the cellular function of the newly discovered DNA damage repair factor WDR70, and investigate the mutation in ovarian cancer to verify if function loss of the WDR70gene was associated with ovarian cancer. METHODS: The WDR70 gene was silenced by using siRNA technique or overexpressed its wild and mutation type by with lentivirus and plasmid in hunman cells. The subcellular localization and biochemical function of WDR70 was analyzes by indirect immunofluorescence and immunoblotting. The expression level of WDR70 and the mutations of its cDNA was checked with RT-PCR sequencing for 1 normal ovarian tissue and 16 ovarian cancer specimen. RESULTS: We found gene silencing of WDR70 or overexpression of WDR70 mutation type disrupts the phosphorylation level of homologous recombination functional protein RPA32 and the ability of recruitment at DNA damage site of recombinase RAD51, the loss of function of WDR70 also causes the elevation of the chromosome breakage in metaphase. Meanwhile, we also noticed that the existence of multiple mutations in genomic WDR70 in ovarian cancer specimen. CONCLUSIONS: Our results defined that in vitro system, WDR70 is a DNA damage repair gene, silencing of WDR70 or overexpression of WDR70 mutation type disrupts homologous recombination and chromosomal instability; the frequent mutations of WDR70 gene in genome of ovarian cancer specimens could also lead to DNA repair defeat and gene instability. Consequently WDR70 gene could represent an anti-cancer mechanism for ovarian cancer.

[DNA damage response of epithelial ovarian cancer cells (primary culture) to chemo-radiotherapy].

OBJECTIVE: To detect protein dynamic changes of cellular localization and the DNA damage response of epithelial ovarian cancer cells to chemo-radiotherapy. METHODS: 28 specimens of epithelial ovarian cancer were collected, with 6 cases diagnosed as borderline serous cystadenoma, 5 as highly differentiated, 6 as medium differentiated and 11 as poorly differentiated cystadenocarcinoma. Collagenase A was used for digesting tissues before primary culture. We compared the characteristics of cells cultured in different mediums (MCDB/M199 medium, primary culture medium, and DMEM medium) supplemented with multiple growth-promoting factors. The characteristics of cells were examined in terms of the maintenance of normal cell morphology, proliferation potential, and cell fibrosis proteins (53BP1 and gamma-H2AX) responsive to DNA damage [those in the ATM checkpoint pathway determined by indirect immunofluorescent staining after treatment with camptothecin (CPT) and X-ray]. RESULTS: Normal morphology was maintained relatively well in the cells cultured in MCDB/M199 medium and its cell fibrosis was slow compared with the cells cultured in other media. Gradually increased endogenous damage was demonstrated by the expression of 53BP1 and gamma-H2AX foci (P < 0.05) in all of the ovarian primary cells. After treatment with CPT and ionizing radiation, increased levels of DNA double-strand breaks were observed indicating a classic DNA damage response. CONCLUSION: We have successfully established a protocol for the primary culture of epithelial ovarian cancer cells, which provides an important platform for characterizing DNA damage responses of the cells. With the progression of epithelial ovarian cancers, the ATM checkpoint pathway is activated by endogenous DNA lesions. This signaling pathway can be further activated by CPT or X-ray irradiation, hampering the growth of tumor cells and further progression of cancers.

[DNA damage response in ovarian clear cell adenocarcinoma].

OBJECTIVE: To study the relationship between ovarian clear cell adenocarcinoma and DNA damage. METHODS: 14 samples were selected from clinical ovarian cases including 3 cases with normal ovarian tissue, 6 cases with poorly differentiated ovarian tumor, 5 cases with ovarian clear cell adenocarcinoma, treated by X-ray irradiation and frozen sections respectively. DNA damage response was analyzed by immunofluorescence and Western blot. RESULTS: Before X-ray irradiation, compared to normal ovarian tissue, a large number of endogenous damage existed in ovarian clear cell adenocarcinoma, and phosphorylation of histone family 2A variant (H2AX) was abnormally enhanced 1 hour after irradiation treatment, however, DNA repair was normal in ovarian clear cell adenocarcinoma. Phosphorylation of H2AX was dispensable for p53 binding protein 1 (53BP1) activation and couldn't be colocalized in clear-type ovarian cancer tissues. CONCLUSION: The abnormal DNA damage activation implies that the network of DNA damage signaling pathway may be defective.

[Clinicopathologic features of 234 cases with borderline ovarian tumors].

OBJECTIVE: To determine the clinicopathologic characteristics and prognostic factors that may be used to predict the poor outcome of patients with borderline ovarian tumors. METHODS: All cases with borderline ovarian tumors treated in the West China Second University Hospital from January 2001 to June 2007 were analyzed retrospectively for clinicopathologic features, treatment parameters and outcome of treatment. Univariate and multivariate analyses were used to assess independent prognostic factors using the logistic regression model. RESULTS: The median age of 234 patients was 40.1 years with a range of 14 to 80 years. There were 101 (43.2%), 94 (40.2%), 19 (8.1%), 12 (5.1%), 8 (3.4%) cases of serous, mucinous, mixed, endometrioid and clear cell tumors, respectively. Out of 234 cases, 182 (77.8%) underwent laparotomy and 45 (19.2%) underwent laparoscopy. Seven women underwent laparoconversion. Fertility sparing surgery was performed on 119 cases (50.9%) and radical surgery was performed on 115 cases (49.1%). Totally 161 (68.8%) patients had stage I, 19 (8.1%) had stage II, 54 (23.1%) had stage III, and none had stage IV disease. Sixty-four women received postoperative chemotherapy. The median follow-up was 40 months with a range of 8 to 78 months. Recurrence was found in 26 cases (11.1%) during follow-up, and no tumor-related death was reported. The logistic regression model showed that surgery procedure (OR = 2.304, P = 0.024), cyst rupture (OR = 2.213, P = 0.038), stage (OR = 4.114, P < 0.01), microinvasion (OR = 2.291, P = 0.046) and peritoneal implants (OR = 2.101, P = 0.016) were the five independent prognostic factors affecting recurrence. CONCLUSIONS: Although patients with borderline ovarian tumors have an excellent prognosis, the risk of recurrence remains in some patients. Emphasis should be put on these patients with high risk factors and preventive strategies should be taken to prevent their progression.

[Improve the chemosensitivity of resistant ovarian cancer cell by small RNA interference].

OBJECTIVE: To investigate the effects of siRNA on the drug resistance reversal of ovarian cancer cell. METHODS: The siRNA was transfected into human ovarian cancer cell line OVCAR8/TR by liposome. ATP-bioluminence assay was applied to measure the drug sensitivity to four chemotherapeutic agents before and after transfection. RESULTS: ATP-bioluminence assay showed that OVCAR8/TR cells were resistant to cDDP, ADM and Taxol. After siRNA transfection, OVCAR8/TR cells were sensitive to ADM and Taxol which are tansported by P-gp. The inhibition rate of ADM was improved from 37% to 58%, and that of Taxol was improved from 26% to 78%. However, the resistance of OVCAR8/TR cells to cDDP was not reversed. CONCLUSION: siRNA can effectively improve the drug resistance to chemotherapeutic agents which are transfered by P-gp. The RNA interference can reverse MDR1-mediated drug resistance in ovarian cancer cell.

[The growth inhibitory effect of non-steroid anti-inflammatory drugs on ovarian cancer].

OBJECTIVE: To evaluate the effects of two commonly used non-steroidal anti-inflammatory drugs (NSAIDs) Celecoxib and Aspirin on the SKOV3 cell growth, apoptosis and neoplasm genesis of ovarian cancer in vitro and vivo. METHODS: The proliferation of SKOV3 cells were determined by MTT assay, the apoptosis of cell were measured by flow cytometry (FCM), and the cell morphologic changes were observed under inverted phase contrast microscope. Xenografted nude mice models of human ovarian cancer were established, and then randomly allocated to treatment or control group, which was administered with either Celecoxib at the dosage of 10, 25, 50 mg/kg or distilled water alone (control) orally daily for 56 d. The mice weight, tumor volume and drug side effects were detected. RESULTS: A dose-dependent inhibition of proliferation of SKOV3 cell appeared after Celecoxib or Aspirin administered for 24 h, and Celecoxib had more inhibiting efficiency to cell growth than Aspirin did. The inhibitory concentration 50% (IC50) in this assay for Celecoxib was 5X 10(-5) mol/L,whereas for Aspirin was 7X 10(-3) mol/L. With cell morphology the "vacuole" presented in the cytoplasm. In contrast to the control, the apoptotic rates (47. 1% and 15. 7%) of SKOV3 were increased after treatment with Celecoxib (5 X 10(-5) mol/L) and Aspirin(7 X 10(-3) mol/L). In nude mice, the average volume of tumor from control mice was (3. 283+/- 0. 432) cm(3) as compared with (2. 457+/- 0. 224) cm(3), (2. 198+/- 0. 500) cm(3), (2. 017+/-0. 166) cm' from Celecoxib mice (10, 25, 50 mg/kg), P<0. 05, and the rates of tumor growth inhibited by 3 Celecoxib dosages to SKOV3 cell burden mice were 25. 20%, 33. 00% and 38. 60%, in a dose-and time-dependent manner, and histopathologic examinations of kidney, liver, stomach and bowel showed no abnormality, with implying no untoward side effects. CONCLUSION: Both COX-2 specific inhibitor Celecoxib and non-selective inhibitor Aspirin can potentially inhibit the tumor growth and induce apoptosis of SKOV3 cells, and the effect of Celecoxib is more potential than that of Aspirin.

[Research on human ovarian cancer cell MDR1 gene silenced by siRNA].

OBJECTIVE: To evaluate the effects of siRNA on the inhibitions of mRNA and P-gp expression of ovarian cancer cell with high expression of multidrug resistance gene (MDR1). METHODS: The siRNA was transferred into ovarian cancer cell line OVCAR8/TR. The real time RT-PCR and flow cytometry were used respectively to determine the expression of mRNA or P-gp of MDR1. RESULTS: The inhibition of mRNA-84% expression occurred at 48 h after cell transfection, and the inhibition of P-gp-85.23% expression occurred at 72 h after cell transfection. Afterward the inhibitions to mRNA and P-gp expressions gradually returned to the normal levels. The amounts of mRNA and P-gp expression had no difference between negative control and untransfected cells. CONCLUSION: RNA interference presents in the human ovarian cancer cell, siRNA can effectively inhibit the expression of mRNA and P-gp of MDR1. The RNAi may represent a new approach for the treatment of MDR1-mediated drug resistance.

[Reversal of multi-drug resistance in ovarian cancer cell by RNA interference].

OBJECTIVE: To investigate the effects of small interference RNA (siRNA) on the inhibition of MDR1 mRNA and P-gp expression of ovarian cancer cells with high expression of MDR1 gene, and reversal of drug resistance. METHODS: siRNA was synthesized and transfected into human ovarian cancer cell line OVCAR8/TR by liposome. The expression of MDR1 mRNA at different times after transfection was measured by real time RT-PCR and the P-gp expression was detected by flow cytometry. Adenosine triphosphate (ATP)-bioluminence assay was applied to check the drug sensitivity to four different chemotherapeutic agents before and after transfection. RESULTS: The suppression rates of MDR1 mRNA were 26.42%, 84.00%, 78.43%, 45.85% and 0 respectively at 24, 48, 72, 96 and 120 hours after transfection. The P-gp suppression rates were 16.71%, 49.64%, 85.23%, 65.98%, 9.44% respectively at 24, 48, 72, 96 and 120 hours after transfection. The maximal suppression rates of MDR1 mRNA and P-gp occurred at 48 and 72 hours after transfection respectively. ATP-bioluminence assay showed that OVCAR8/TR cells were sensitive to fluorouracil, resistant to cisplatin, doxorubicin (adriamycin) and paclitaxel (taxol). After siRNA treatment, OVCAR8/TR cells were sensitive to paclitaxel and doxorubicin, but the resistance to cisplatin could not be reversed. CONCLUSIONS: RNA interference (RNAi) presents in human ovarian cancer cells. siRNA can effectively inhibit the expression of mRNA and P-gp of the multidrug resistance gene MDR1, and can reverse the drug resistance to chemotherapeutic agents which are transferred by P-gp.

[Effects of nonsteroidal anti-inflammatory drug celecoxib on expression of cyclooxygenase-2 (COX-2) in ovarian carcinoma cell].

OBJECTIVE: To explore the effects of nonsteroidal anti-inflammatory drug Celecoxib on the expression of cyclooxygenase-2 (COX-2) in the SKOV3 cell line and the xenografted nude mice of ovarian carcinoma. METHODS: The expression of COX-2 in the SKOV3 cell was determined by reverse transcription polymerase chain reaction (RT-PCR), flow cytometry (FCM), and Western blot analysis. The expression of COX-2 in tumor cells was measured with Immunocytochemistry. RESULTS: RT-PCR showed that the expression COX-2 mRNA was strongly down-regulated in SKOV3 cells after treatment with Celecoxib or Aspirin. FCM and Western blot analysis showed that the protein product of COX-2 was strongly decreased by Celecoxib or Aspirin. The Celecoxib was more potential effects than Aspirin. The immunocytochemistry result showed that the expression of COX-2 in 10, 25, 50 mg/kg x d of Celecoxib were lower obviously than it in the control group in Xenografted nude mice. CONCLUSION: The anticarcinogenic effects of Celecoxib is probably related to the down-regulation of COX-2, and can be explained to both COX-2-dependent and -independent mechanisms.

Decreased histone H2B monoubiquitination in malignant gastric carcinoma.

AIM: To investigate H2B monoubiquitination (uH2B) and H3K4 di- and tri-methylation (H3K4-2me, H3K4-3me) levels and their clinical significance in gastric cancer (GC). METHODS: Immunohistochemistry (IGC) was used to detect the differential levels of uH2B, H3K4-2me and H3K4-3me modifications in GC specimens from chemo/radiotherapy-naïve patients who underwent potentially curative surgical resection (n = 159) and in a random sampling of non-tumor gastric epithelium specimens (normal controls, n = 20). The immunohistochemistry (IHC)-detected modifications were classified as negative, low-level, or high-level using a dual-rated (staining intensity and percentage of positively-stained cells) semi-quantitative method. The relationships between uH2B modification levels and clinicopathological parameters of GC were assessed by a Wilcoxon rank sum test (pairwise comparisons) and the Kruskal-Wallis H test (multiple comparisons). The correlation between uH2B modification and survival was estimated by Kaplan-Meier analysis, and the role of uH2B as an independent prognostic factor for survival was assessed by multivariate Cox regression analysis. RESULTS: The presence and level of H3K4-2me and H3K4-3me IHC staining was similar between the normal controls and GC specimens. In contrast, the level of uH2B was significantly lower in the malignant gastric tissues (vs normal control tissues) and decreased along with increases in dedifferentiation (well differentiated > moderately differentiated > poorly differentiated). The level of uH2B correlated with tumor differentiation (P < 0.001), Lauren's diffuse- and intestinal-type classification (P < 0.001), lymph node metastasis (P = 0.049) and tumor-node-metastasis stage (P = 0.005). Patients with uH2B+ staining had higher 5-year survival rates than patients with uH2B-staining (52.692 ± 2.452 vs 23.739 ± 5.207, P < 0.001). The uH2B level was an independent prognostic factor for cancer-specific survival (95%CI: 0.237-0.677, P = 0.001). CONCLUSION: uH2B displays differential IHC staining patterns corresponding to progressive stages of GC. uH2B may contribute to tumorigenesis and could be a potential therapeutic target.

[Detection of physical status of human papillomavirus 16 in cervical cancer tissue and SiHa cell line by multiplex real-time polymerase chain reaction].

BACKGROUND & OBJECTIVE: The integration of high-risk human papillomavirus (HPV) into host cell genome is one of the major contributing factors to cervical malignant transformation. The detection of HPV integration is helpful for understanding its role in cervical carcinogenesis and tumor progression. However, there is no ideal detection method of HPV physical status for clinical use. The study was to explore an ideal method of detecting the physical status of HPV-16. METHODS: Setting HPV-16 plasmid as standard, multiplex real-time polymerase chain reaction (PCR) using 2 different fluorescent report radicals was used to quantify the copy numbers of E2 and E6 genes for analysis of the physical status of HPV-16 according to E2/E6. Multiplex real-time PCR test and Southern blot results of cervical cancer cell line SiHa and 27 specimens of HPV-16-positive cervical squamous cell carcinoma were compared. RESULTS: There was a linear relationship between the threshold cycle values and the copy numbers of E2 and E6 in both standard curves, with the correlation coefficients of 1.00 and the amplification efficiencies of above 95%. The 95% reference range of plasmid E2/E6 ratio, in which the amount of E2 DNA was equal to that of E6 DNA, was 0.81-1.29. The cut-off value of E2/E6, which was used to distinguish the pure episomal form from a mixed form of episomal and integrated HPV-16, was 0.81 in the multiplex real-time PCR test. HPV-16 was observed to be integrated into the host genome of SiHa cells by multiplex real-time PCR and Southern blot. The coincidence rate of multiplex real-time PCR and Southern blot was 81.5% (22/27) in the cervical squamous cell cancer tissues (kappa=0.844, P<0.001). CONCLUSION: Multiplex real-time PCR test is a rapid, sensitive and reliable method for detecting the physical status of HPV-16 DNA, and is convenient to be applied in paraffin-embedded tissue and small preneoplastic or early neoplastic cervical lesions, even in cervical scrapes which contain a small amount of DNA.