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The "Supplemental Data" contains supplementary methods,supplementary table 1 and supplementary figures1-19.1.Supplementary methods: Antibodies and Reagents, Primary culture of LECs, Immunomagnetic isolation of LECs, Expressing human recombinant sSEMA4C protein, Liquid chromatography - Electrospray ionization tandem MS (LC-ESI/MS), Migration assay, Wound healing assay, Cell proliferation assay, Primers sequences; 2.Supplementary Table 1: the Patient characteristics; 3.Supplementary Figure 1: the Authentication of LECs; 4.Supplementary Figure 2: the Evaluation of LCM-isolated LECs using RNA markers; 5. Supplementary Figure 3 : SEMA4C expresses on lymphatic vessels; 6.Supplementary Figure 4 : Nano-LC-ESI-MS/MS spectrum of a single charged deglycosylated glycopeptide from the culture supernatants of tumor LECs; 7. Supplementary Figure 5 :sSEMA4C binds to tumor cells and LECs; 8.Supplementary Figure 6: sSEMA4C promotes the migration and proliferation of tumor cells; 9.Supplementary Figure 7: SEMA4C promotes LECs function in vitro; 10.Supplementary Figure 8 : targeting SEMA4C inhibits LECs function in vitro; 11.Supplementary Figure 9 : the growth of the tumors; 12.Supplementary Figure 10 : Knock-down of PlexinBs; 13.Supplementary Figure 11 : Blocking ERBB2 or PlexinB2 inhibits SEMA4C-induced lymphangiogenesis in vitro; 14.Supplementary Figure 12 : LEC was stimulated by VEGF-C and sSEMA4C; 15.Supplementary Figure 13 : sSEMA4C activates MET and RHOA in tumor cells; 16.Supplementary Figure 14 : blocking MET or PlexinB2 inhibits SEMA4C-induced migration and proliferation of tumor cells; 17.Supplementary Figure 15 : cells treated with sSEMA4C; 18.Supplementary Figure 16 : targeting RHOA inhibits SEMA4C-induced lymphangiogenesis in vitro and in vivo; 19.Supplementary Figure 17 : targeting RHOA inhibits SEMA4C-induced migration of tumor cells; 20.Supplementary Figure 18 : 3 days after inoculation with tumour cells, the mice were treated with Lapatinib, K252a ,C3 toxin, Fasudil, Y27632 , sSEMA4C and PBS control solution, the size of tumor was measured; 21.Supplementary Figure 19: 3 days after inoculation with tumour cells, the mice were treated with Lapatinib, K252a ,C3 toxin, Fasudil, Y27632 , sSEMA4C and PBS control solution, the average number of positive lymph node in each mice were calculated.

Purpose: Lymphatic vessels are mainly regarded as passive conduits for the dissemination of cancer cells. In this study, we investigate whether and how the tumor-associated lymphatic vessels may play an active role in tumor metastasis.Experimental Design:In situ laser capture microdissection of lymphatic vessels followed by cDNA microarray analysis was used to determine the expression profiling of lymphatic endothelial cells (LEC). Gene expression levels and activity of signaling pathways were measured by real-time RT-PCR, ELISA, or immunoblotting. Lymphangiogenesis was assessed by IHC. Lymph node metastasis was measured using fluorescence imaging. The effects of SEMA4C on lymphangiogenesis in vitro were evaluated using migration assay and tube-formation assay of LECs.Results: Tumor-associated LECs are molecularly and functionally different from their normal counterparts. In addition to expressing high levels of membrane-bound SEMA4C, tumor-associated LECs also produced soluble SEMA4C (sSEMA4C). Increased serum sSEMA4C was detected in patients with breast cancer and cervical cancer. Patients with metastasis had much higher levels of serum sSEMA4C. sSEMA4C promoted lymphangiogenesis by activating PlexinB2-ERBB2 signaling in LECs, and promoted the proliferation and migration of tumor cells by activating PlexinB2-MET signaling, thus promoting lymphatic metastasis. Although the SEMA4C signaling pathways differ between LECs and tumor cells, RHOA activation was necessary for the effects of SEMA4C in both types of cells.Conclusions: Tumor-associated LECs produce sSEMA4C to promote lymphatic metastasis of tumors. Our results suggest that SEMA4C and RHOA might be potential therapeutic targets, and that higher serum sSEMA4C could be a marker for breast cancer and cervical cancer. Clin Cancer Res; 23(1); 214–24. ©2016 AACR.

Supplementary Methods and Supplementary Figure 1-20: Supplementary Figure 1 shows the growth of the tumors after orthotopic transplantation. Supplementary Figure 2 shows that over-expression of SIX1 promotes lymphangiogenesis and lymph-node metastasis of C33a cells in vivo. Supplementary Figure 3 shows the assay of cytokine and growth factor with antibody-based array. Supplementary Figure 4 shows the expression of the genes coding for the lymphangiogenic factors. Supplementary Figure 5 shows that SIX1 promotes VEGF-C expression in C33a cells. Supplementary Figure 6 shows that SIX1 promotes migration and tube-formation of HLECs. Supplementary Figure 7 shows the knock-down of VEGF-C in cervical cancer cells. Supplementary Figure 8 shows that the promotional effect of SIX1-expressing tumor cells on the migration and tube-formation of HLECs in vitro is mediated by VEGF-C. Supplementary Figure 9 shows that the promotional effect of SIX1-expressing tumor cells on lymphangiogenesis in vivo is mediated by VEGF-C. Supplementary Figure 10 shows that the promotional effect of SIX1-expressing C33a cells on lymphatic-vessel is mediated by VEGF-C. Supplementary Figure 11 shows that the promotional effect of SIX1 on lymph-node metastasis in vivo is mediated by VEGF-C. Supplementary Figure 12 shows the differences in gene expression levels between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 13 shows the immunohistochemical analysis of TGF-βs in tissue microarray of human CSCC specimens. Supplementary Figure 14 shows that SIX1 enhances the effect of TGF-β1 on VEGF-C production by tumor cells and tube-formation of HLECs in vitro. Supplementary Figure 15 shows that TGF-β contributes to the differences in VEGF-C expression between tumors in vivo and tumor cells cultured in vitro. Supplementary Figure 16 shows that TGF-β signaling is required for SIX1 to promote lymphangiogenesis and lymph-node metastasis in vivo. Supplementary Figure 17 shows that knocking down the expression of SMAD2 or SMAD3 or TβR1 suppresses TGF-β1 induced VEGF-C expression. Supplementary Figure 18 shows that higher expression of SIX1 correlates with higher expression of TβR1 in human CSCC specimens. Supplementary Figure 19 shows the binding of SIX1 to the VEGF-C promoter. Supplementary Figure 20 shows that knocking down the expression of SMAD2 and SMAD3 does not affect the binding of SIX1 to the VEGF-C promoter.

Platelets are reprogrammed by cancer via a process called education, which favors cancer development. The transcriptional profile of tumor-educated platelets (TEPs) is skewed and therefore practicable for cancer detection. This intercontinental, hospital-based, diagnostic study included 761 treatment-naïve inpatients with histologically confirmed adnexal masses and 167 healthy controls from nine medical centers (China, n = 3; Netherlands, n = 5; Poland, n = 1) between September 2016 and May 2019. The main outcomes were the performance of TEPs and their combination with CA125 in two Chinese (VC1 and VC2) and the European (VC3) validation cohorts collectively and independently. Exploratory outcome was the value of TEPs in public pan-cancer platelet transcriptome datasets. The AUCs for TEPs in the combined validation cohort, VC1, VC2, and VC3 were 0.918 (95% CI 0.889–0.948), 0.923 (0.855–0.990), 0.918 (0.872–0.963), and 0.887 (0.813–0.960), respectively. Combination of TEPs and CA125 demonstrated an AUC of 0.922 (0.889–0.955) in the combined validation cohort; 0.955 (0.912–0.997) in VC1; 0.939 (0.901–0.977) in VC2; 0.917 (0.824–1.000) in VC3. For subgroup analysis, TEPs exhibited an AUC of 0.858, 0.859, and 0.920 to detect early-stage, borderline, non-epithelial diseases and 0.899 to discriminate ovarian cancer from endometriosis. TEPs had robustness, compatibility, and universality for preoperative diagnosis of ovarian cancer since it withstood validations in populations of different ethnicities, heterogeneous histological subtypes, and early-stage ovarian cancer. However, these observations warrant prospective validations in a larger population before clinical utilities.

ObjectivesAdvancements in big data technology are reshaping the healthcare system in China. This study aims to explore the role of medical big data in promoting digital competencies and professionalism among Chinese medical students.Design, setting and participantsThis study was conducted among 274 medical students who attended a workshop on medical big data conducted on 8 July 2021 in Tongji Hospital. The workshop was based on the first nationwide multifunction gynecologic oncology medical big data platform in China, at the National Union of Real-World Gynecologic Oncology Research & Patient Management Platform (NUWA platform).Outcome measuresData on knowledge, attitudes towards big data technology and professionalism were collected before and after the workshop. We have measured the four skill categories: doctor‒patient relationship skills, reflective skills, time management and interprofessional relationship skills using the Professionalism Mini-Evaluation Exercise (P-MEX) as a reflection for professionalism.ResultsA total of 274 students participated in this workshop and completed all the surveys. Before the workshop, only 27% of them knew the detailed content of medical big data platforms, and 64% knew the potential application of medical big data. The majority of the students believed that big data technology is practical in their clinical practice (77%), medical education (85%) and scientific research (82%). Over 80% of the participants showed positive attitudes toward big data platforms. They also exhibited sufficient professionalism before the workshop. Meanwhile, the workshop significantly promoted students’ knowledge of medical big data (p<0.05), and led to more positive attitudes towards big data platforms and higher levels of professionalism.ConclusionsChinese medical students have primitive acquaintance and positive attitudes toward big data technology. The NUWA platform-based workshop may potentially promote their understanding of big data and enhance professionalism, according to the self-measured P-MEX scale.

Background: Morpho-physiological alternations of platelets provided a rationale to harness RNA sequencing of tumor-educated platelets (TEPs) for preoperative diagnosis of cancer. Timely, accurate, and non-invasive detection of ovarian cancer in women with adnexal masses presents a significant clinical challenge. Patients and Methods: This intercontinental, hospital-based, diagnostic study included 761 treatment-naïve inpatients with histologically confirmed adnexal masses and 167 healthy controls from nine medical centers (China, n=3; Netherlands, n=5; Poland, n=1) between September 2016 and May 2019. The main outcomes were the performance of TEPs and their combination with CA125 in two Chinese (VC1 and VC2) and the European (VC3) validation cohorts collectively and independently. Exploratory outcome was the value of TEPs in public pan-cancer platelet transcriptome datasets. Results: The AUCs for TEPs in the combined validation cohort, VC1, VC2, and VC3 were 0.918 (95% CI 0.889-0.948), 0.923 (0.855-0.990), 0.918 (0.872-0.963), and 0.887 (0.813-0.960), respectively. Combination of TEPs and CA125 demonstrated an AUC of 0.922 (0.889-0.955) in the combined validation cohort; 0.955 (0.912-0.997) in VC1; 0.939 (0.901-0.977) in VC2; 0.917 (0.824-1.000) in VC3. For subgroup analysis, TEPs exhibited an AUC of 0.858, 0.859, and 0.920 to detect early-stage, borderline, non-epithelial diseases and 0.899 to discriminate ovarian cancer from endometriosis. Analysis of public datasets suggested that TEPs had potential to detect multiple malignancies (Table 1). Conclusions: TEPs had robustness, compatibility, and universality for preoperative diagnosis of ovarian cancer since it withstood validations in populations of different ethnicities, heterogeneous histological subtypes, early-stage ovarian cancer as well as other malignancies. However, these observations warrant prospective validations in a larger population before clinical utilities. Table 1. Performance for TEPs in public pan-cancer datasets. Disease n Healthy Control AUC, area under the curve (95% CI) Women NSCLC (non-small-cell lung cancer) 126 77 0.758 (0.691-0.825) Breast cancer 38 77 0.817 (0.726-0.909) Colorectal cancer 18 77 0.973 (0.945-1.000) Pancreatic cancer 16 77 0.993 (0.981-1.000) Glioblastoma 10 77 0.923 (0.831-1.000) Men NSCLC 119 82 0.746 (0.677-0.815) Colorectal cancer 25 82 0.933 (0.884-0.982) Pancreatic cancer 22 82 0.993 (0.984-1.000) Glioblastoma 19 82 0.981 (0.959-1.000) All NSCLC 245 159 0.774 (0.728-0.820) Colorectal cancer 40 159 0.978 (0.961-0.996) Breast cancer 38 159 0.821 (0.736-0.906) Pancreatic cancer 35 159 0.987 (0.974-0.999) Glioblastoma 35 159 0.931 (0.890-0.972) Hepatobiliary carcinomas 14 159 0.991 (0.978-1.000) Citation Format: Yue Gao, Chun-Jie Liu, Hua-Yi Li, Xiao-Ming Xiong, Sjors G.j.g. In ‘t Veld, Gui-Ling Li, Jia-Hao Liu, Guang-Yao Cai, Gui-Yan Xie, Shao-Qing Zeng, Yuan Wu, Jian-Hua Chi, Qiong Zhang, Xiao-Fei Jiao, Lin-Li Shi, Wan-Rong Lu, Wei-Guo Lv, Xing-Sheng Yang, Jurgen M.j. Piek, Cornelis D de Kroon, C.a.r. Lok, Anna Supernat, Sylwia Łapińska-Szumczyk, Anna Łojkowska, Anna J. Żaczek, Jacek Jassem, Bakhos A. Tannous, Nik Sol, Edward Post, Myron G. Best, Bei-Hua Kong, Xing Xie, Ding Ma, Thomas Wurdinger, An-Yuan Guo, Qing-Lei Gao. Platelet RNA signature enables early and accurate detection of ovarian cancer: An intercontinental, biomarker identification study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB168.

e17578 Background: Olaparib demonstrated its efficacy to prolong progression-free survival (PFS) in platinum-sensitive relapsed ovarian cancer (PSROC) patients (L-MOCA study). Almost half of OC patients harbor homologous recombination deficiencies (HRD). However, there is lack of the HRD prevalence in Asian PSR population. Meanwhile, Immunological processes play a key role in tumorigenesis and PD-L1 status may hinder an effective antitumor immune response in OC. Herein, we analyzed HRD and PD-L1 status in Asian patients with PSROC, and explored Olaparib response to different HRD status. Methods: In this exploratory analysis of L-MOCA trial, HRD and PD-L1 status were analyzed from patients with PSROC. Residual DNA samples of 193 patients were analyzed for HRD status with a local developed HRD assay (Teddy Laboratory, Shanghai, China, which determines two major components, tBRCA1/2 status and LOH score, HRD positive indicates the presence of a deleterious or suspected deleterious BRCA mutation or LOH score ≥0.4). The remaining formalin fixation and paraffin embedded samples were used for PD-L1 expression analysis by immunohistochemistry with SP263. Descriptive statistics were summarized for HRD status, PD-L1 expression status and PFS by different HRD status subgroups. Results: 193 of 224 patients had residual DNA for HRD test. Patients clinical characteristics were similar between those with and without residual DNA for HRD test, in terms of age (mean age 55.5y Vs 54.2y), FIGO stage (stage III 68.9% Vs 61.3%, stage IV 12.4% Vs 22.65%), and time to disease progression to Last prior Platinum Based Chemotherapy (6-12m: 39.9% Vs 41.9%; > 12m: 59.6% Vs 54.8%). Among 193 patients with HRD test conducted, proportion of HRD+ BRCAm, HRD+ BRCAwt, HRD- and HRD unknown were 37.3%, 28.0%, 14.5% and 20.2%, separately. mPFS of the above subgroups was 20.1 months, 15.8 months, 9.2 months and 16.4 months, respectively (Table1). 200 of 224 patients were available for PD-L1 expression analysis. 184 (92%) with TC＜1%, 12 (6%) with TC among 1-25%. These results showed TC expression in all patients were less than 25%, suggesting that ovarian cancer may be an immunologically cold tumor. Conclusions: This is firstly showing the HRD and PD-L1 status in Asian OC patients. The results showed all the PSR patients could benefit from Olaparib treatment, regardless of HRD status. Meanwhile the numerically different mPFS observed across different HRD status subgroups also indicated potential clinical utility of locally developed HRD assay. Clinical trial information: NCT03534453. [Table: see text]

BackgroundOvarian clear cell carcinoma (OCCC) has an abysmal prognosis with a median overall survival (OS) of 25.3 months because of a low response to chemotherapy. The 5-year disease-specific survival rate after recurrence is 13.2%, with more than two-thirds of the patients dying within a year. Therefore, it is urgent to explore new therapeutic options for OCCC. Based on the characteristic immune-suppressive tumour microenvironment derived from the gene expression profile of OCCC, the combination of immunoantiangiogenesis therapy might have certain efficacy in recurrent/persistent OCCC. This trial aims to evaluate the efficacy and safety of sintilimab and bevacizumab in patients who have failed platinum-containing chemotherapy with recurrent or persistent OCCC.Method and analysisIn this multicentre, single-arm, open-label, investigator-initiated clinical trial, 38 patients will be assigned to receive sintilimab 200 mg plus bevacizumab 15 mg/kg every 3 weeks. The eligibility criteria include histologically diagnosed patients with recurrent or persistent OCCC who have been previously treated with at least one-line platinum-containing chemotherapy; patients with Eastern Cooperative Oncology Group (ECOG) performance status 0–2 with an expected survival greater than 12 weeks. The exclusion criteria include patients previously treated with immune checkpoint inhibitor and patients with contraindications of bevacizumab and sintilimab. The primary endpoint is the objective response rate. The secondary endpoints are progression-free survival, time to response, duration of response, disease control rate, OS, safety and quality of life. Statistical significance was defined as pEthics and disseminationThis trial was approved by the Research Ethics Commission of Tongji Medical College of Huazhong University of Science and Technology (2020-S337). The protocol of this study is registered at www.clinicaltrials.gov. The trial results will be published in peer-reviewed journals and at conferences.Trial registration numberNCT04735861; Clinicaltrials. gov.

Epithelial-mesenchymal transition (EMT) plays a critical role in promoting tumor invasion and metastasis. However, the key cofactors that modulate the signal transduction to induce EMT have note been fully explored to date. The present study reports that sine oculis homeobox homolog 1 (SIX1) is able to promote EMT of cervical cancer by coordinating with transforming growth factor (TGF)β-SMAD signals. The expression of SIX1 was negatively correlated with the expression of the epithelial marker E-cadherin in two independent groups of cervical cancer specimens. SIX1 could promote the transition of mesenchymal phenotype in the presence of active TGFβ signals in vitro and in vivo. TGFβ-SMAD signals were required for the SIX1-mediated promotion of EMT and metastatic capacity of cervical cancer cells. Together, SIX1 and TGFβ cooperated to induce more remarkable changes in the transition of phenotype than each of them alone, and coordinated to promote cell motility and tumor metastasis in cervical cancer. These results suggest that the coordination of SIX1 and TGFβ signals may be crucial in the EMT program, and that SIX1/TGFβ may be considered a valuable marker for evaluating the metastatic potential of cervical cancer cells, or a therapeutic target in the treatment of cervical cancer.

The concept of intelligent manufacturing influences the revolution of manufacture industry deeply, and makes enterprises require more from their employees. Facing the new trend of intelligent manufacturing, the changes of posts and talent demands should be deeply analyzed and the talents training mode should be optimized continuously to meet the changes.

With increasing global climate change, analyses of stress-inducing conditions have important significance in ecological adaptation and the biological distribution of species. To reveal the difference in temperature-adaptive strategy between Turpan and Beijing populations of Bemisia tabaci (Gennadius) Middle East Asia Minor 1 (MEAM1) under high-temperature stress conditions, we compared thermal tolerance and life history traits between Beijing and Turpan populations of MEAM1 after exposure to different heat shock treatments for different times. The experimental design reflected the nature of heat stress conditions suffered by MEAM1. The results showed that eggs, red-eyed pupae, and adults of the Turpan population were more heat tolerant than those of the Beijing population under the same stress conditions. Additionally, it was found that longevity and F1 adult survival rate were significantly higher in the Turpan population than in the Beijing population after heat shock stress, but egg number and F1 female ratio were not significantly different between Turpan population and Beijing population. Overall, it was suggested that heat tolerance and longevity traits were the most relevant for climate characteristics and not reproductive traits, and improved heat tolerance and prolonged longevity were important adaptive strategies that helped MEAM1 to survive in harsh high-temperature conditions such as Turpan arid desert climate. The present results provided further insight into the modes of heat tolerance and the ways in which survival and longevity traits respond to environmental selection pressures.

To investigate the changes in cell cycle induced by cisplatin (DDP) and the effect of antisense oligonucleotide (AsODN) targeting Chk1/2 on DDP-induced apoptosis in lung cancer cell line A549 cells.The characteristics of cell cycle and apoptosis induced by DDP were detected by flow cytometry using SubG1 method. Chk1/2 mRNA and protein expression were assayed by RT-PCR and Western blot under best condition of transfection of AsODN targeting Chk1/2 by lipofection. Apoptosis of A549 cells induced by DDP was determined by flow cytometry using AnnexinV-FITC staining after transfection of Chk1/2 AsODN.Asynchronized A549 cells were treated with 10 micromol/L DDP, and significant S-phase arrest was observed at 12 h later. Transfection with antisense oligonucleotide targeting Chk1/2 inhibited the Chk1/2 expression at both mRNA and protein levels. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 100% - 200%, compared with that in the sODN control (P0.05), but combined use of Chk1- and Chk2-specific AsODN did not show synergistic effects as compared with that induced by treatment with Chk1- or Chk2-specific AsODN alone (P0.05).Chk1 and Chk2 may be regarded as effective targets of chemotherapy for lung cancer. Silencing the key effector Chk1 and Chk2 genes may significantly increase the chemosensitivity of lung cancer cells.

To explore the increasing effect of blocking Chk1 and /or Chk2 gene by Chk1 or Chk2-specific antisense oligodeoxynucleotides (AsODN) on apoptosis in HeLa cell line after irradiation and its mechanism of action.Asynchronized HeLa cells were exposed to (60)Co-irradiation at different dosage to activate G(2)/M checkpoint arrest. The cell cycle profiles were observed in HeLa cells after irradiation at a range of various doses and different time points by flow cytometry. In the experimental groups, Chk1/2 sODN and AsODN alone or in combination were transfected into HeLa cells, and the cells were exposed to (60)Co-irradiation at 24 h after transfection. The changes of Chk1/2 protein expression were assayed by Western blot and confocal laser scanning microscopy (Confocal), and the cell cycles, apoptosis rates and cell cycle specific apoptosis were detected by annexin V-PI labeling and flow cytometry.Apoptotic response was significantly increased in the Hela cells after G(2)/M arrest and was inversed to activation of G(2)/M checkpoint. Either Chk1 or Chk2-specific AsODN consistently enhanced DNA damage-induced apoptosis by 90% approximately 120%, compared to corresponding sODN control (P0.05). Unexpectedly, combined use of Chk1- and Chk2-specific AsODN did not produce synergistic effect as compared to treatment with Chk1- or Chk2-specific AsODN alone (P0.05). While irradiated HeLa cells underwent apoptosis preferentially in G(1)-phase, apoptosis occurred in either of G(1)-, S- or G(2)/M -phase in the presence of Chk1 and/or Chk2 AsODN.The radioresistance is mainly induced by activating the cell cycle checkpoint signal transduction pathway after irradiation, and abrogating of the key effector Chk1 and Chk2 may increase the apoptotic sensitivity to irradiation due to changes of the pattern of cell cycle specific apoptosis.

Replicating adenovirus, with the favorable features of efficient transduction and multiple in malignant cells, represent a promising option for gene therapy. Its influence on vascular endothelia in vivo is an important indicator for safety evaluation. This study was to explore the effects of conditionally replicating adenovirus Adv5/dE1A-Aschk1 on proliferating and resting endothelial cells.Human umbilical vein endothelial cells (HUVECs) were isolated by filling umbilical veins with digestive enzyme solution. HUVECs were cultured and observed with immunofluorescent staining and under electron microscope to identify endothelial cells. The oncolytic effects of Adv5/dE1A-Aschk1 on HeLa and endothelial cells were measured by cytopathic effect assay (CPE)û its effect on the proliferation of endothelial cells was measured by MTT assay. Cell apoptosis after treatment of Adv5/dE1A-Aschk1 alone or in combination with irradiation was measured by flow cytometry (FCM).Highly purified endothelial cells were isolated. Adv5/dE1A-Aschk1 lysed HeLa cells in a replication-dependent fashion, but did not exhibit detectable cytopathic effects on resting endothelial cells at a multiplicity of infection (MOI) of 500. The inhibitory effect of Adv5/dE1A-Aschk1 on the proliferation of endothelial cells increased with the increase of viral titer. When treated with Adv5/dE1A-Aschk1 at a MOI of 100 for 96 h, the proliferation inhibition rate of proliferating HUVECs was (22.0+/-2.5)%, but that of resting HUVECs was (13.0+/-2.3)%. When treated with Adv5/dE1A-Aschk1 combined irradiation, the apoptosis rate of resting HUVECs was (23.1+/-2.5)%, and that of proliferating HUVECs was (35.7+/-3.0)%.HUVECs may be taken as a good target for the research of conditionally replicating adenovirus. The viral titer for injuring endothelial cells is much higher than that for killing tumor cells, so Adv5/dE1A-Aschk1 has a wide range of therapeutic dosage. As compared with resting HUVECs, proliferating HUVECs are more likely to be lysed by Adv5/dE1A-Aschk1.

Ku80 is a key protein plays a role in repairing DNA double strand break (DSB) after irradiation. There are a few studies about other roles of Ku80 except DSB repair. This study was to inhibit Ku80 expression in cervical carcinoma cell line HeLa with small interfering RNA (siRNA), and explore its effect on cell proliferation.Plasmids pKu80-siRNA and pNeg-siRNA (negative control) were constructed and transfected into HeLa cells. The expression of Ku80 in HeLa cells was detected by Western blot. The proliferation of HeLa cells in vitro and in vivo was determined by clone formation assay, MTT assay, and subcutaneous tumor formation in nude mice.Two cell clones were screened from pKu80-siRNA-and pNeg-siRNA-transfected HeLa cells. Ku80 expression in HeLa cells was suppressed markedly after transfection of pKu80-siRNA; this clone was named Hela/Ku80-siRNA. The clone formation efficiency was significantly lower in HeLa/Ku80-siRNA cells than in control cells (0.46+/-0.05 vs. 0.62+/-0.02, t=5.11, P0.01). The proliferation rate was significantly lower in HeLa/Ku80-siRNA cells than in control cells at 48 h and 72 h after transfection (P0.05). At the 25th day after subcutaneous transplantation in nude mice, the tumor volume was significantly smaller in HeLa/Ku80-siRNA group than in control group [(18.92+/-3.60) mm(3) vs. (194.88+/-30.61) mm(3), t=12.69, P0.01].We successfully established a cell model that Ku80 expression is suppressed almost completely by siRNA. Ku80 inhibition inhibits the proliferation of HeLa cells in vivo and in vitro.

Ataxia telangiectasis is caused by the mutation of AT gene (ATM) and it is characterized by hypersensitivity to the radiation. In order to investigate the relationship between ATM mRNA expression of K562 and SiHA two kinds of tumor cell lines and their cell cycle restardation after gamma-irradiation, their ATM mRNA expressions were measured by semi-quantitive RT-PCR and the cells were irradiated at the dose of 6, 10 and 15 Gy of (60)Co gamma ray and the change of the apoptosis and cell cycle arrest phenomenon were observed at the time of 6, 12, 24, 48 and 60 hours after irradiation. The results showed that the ATM mRNA relative expression level of K562 cell line was 0.04, that of SiHA cell line was 0.80, the ATM transcript levels in SiHA cells were 20 times as much as that in K562. In conclusion, the G(2)/M phase restardation after irradiation was observed in both cell lines, whereas SiHA exhibited a much stronger cell cycle restardation, a self-protection function, than that of K562.

Chemokine receptors express on many tumor cells, and closely correlate with migration and metastasis of tumor cells. This study was to investigate expressions of chemokine(C-X-C) receptor 4 (CXCR4) and chemokine (C-X-C motif) ligand 12 (CXCL12) in human ovarian epithelial tumor cells, and their effects on migration of tumor cells.Expression of CXCR4 mRNA and protein in 15 specimens of epithelial ovarian cancer tissue, ovarian cancer cell line CAOV3, endothelial cell line HUVEC, and 10 specimens of normal ovary tissue were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Expression of CXCL12 mRNA in retroperitoneal lymph nodes, and smooth muscle of fallopian tube from the same 15 epithelial ovarian cancer patients was tested by RT-PCR, quantity of CXCL12 in ascites of 15 patients was assayed using ELISA. Boyden Transwells was used to analyze effects of CXCL12, and cancerous ascites on chemotaxis of CAOV3, and HUVEC cells.(1) Expression levels of CXCR4 mRNA in ovary cancer tissues, CAOV3 cells, and HUVEC cells were 2.30+/-1.12, 1.89+/-1.20, and 1.68+/-1.11, respectively; those of CXCR4 protein were 1.35+/-0.14, 1.86+/-0.34, and 1.96+/-0.23, respectively; CXCR4 mRNA and protein can't be detected in normal ovarian tissues. (2) In 15 ovarian cancer patients, concentrations of CXCL12 in ascites were 632-9 326 pg/ml, and CXCL12 mRNA level in retroperitoneal lymph nodes was 1.14+/-0.87, CXCL12 mRNA can't be detected in smooth muscle of fallopian tube. (3) Recombinant human CXCL12 induced migration of CAOV3, and HUVEC cells, the chemotactic indices (CI) were 3.9+/-1.2, and 4.1+/-1.6, significantly higher than those of control (1.0+/-0.4, and 1.1+/-0.7) (P0.05)u cancerous ascites induced migration of CAOV3 cells with CI of 1.9+/-0.8, significantly higher than that of control (P0.05).CXCR4 and CXCL12 may play roles in metastasis of epithelial ovarian cancer by promoting migration of tumor cells and endothelial cells.

Metastasis is the leading cause of treatment failure and death of ovarian cancer. However, The molecular mechanisms associated with acquisition of metastatic ability in ovarian cancer are poorly understood. This study aimed at selecting the ovarian carcinoma cell lines with high frequency metastasis and studing the association between nm23-H1 gene expression in the model of ovarian carcinoma cells so as to provide the evidence for systematical experimental studying and clinical practice.Each ovarian cancer cell line was transplanted subcutaneously into the flank of nude mice, and the metastatic behavior was evaluated by counting the number of lung tumor foci at different time. The metastatic tumors were cultured in vitro, then established substrain and transplanted subcutaneously three times. The mRNA and protein level of nm23 in 8 human ovarian cancer cell lines were examined.Four cell lines have high frequency metastatic potentiality. The subpopulation of cells with high frequency metastasis could be screened by injection several times. The expression of nm23 mRNA and protein in human ovarian cancer cells is inversely related to metastatic behavior in experimental animals (r=0.96, P=0.0001).The difference of metastatic potential, which was determined by genetic and molecular levels, was significant among different type of cell lines and subtypes. The expression of nm23 mRNA and protein in human ovarian carcinomas were correlated closely with the reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator of ovarian cancer.

To explore the feasibility of reverse effect of recombinant nm23H(1) adeno-associated virus (rAAV-nm23H(1)) in ovarian carcinoma metastatic orthotopic implantation nude model.Using DNA recombination technique to construct AAV main plasmid pUF(1)-nm23H(1). rAAV-nm23H(1) and rAAV-Laz were produced by co-transfection of rAAV system in package cell 293 by phosphate-calcium deposit method, and then the transfection efficiency was measured. The titer was measured by dot hybridization. Ovarian carcinoma cell line SW626 was used in the establishment of the ovarian carcinoma orthotopic implantation nude model. The biologic feature of the model was observed and the expression of nm23H(1) in the tumor was measured by Western blot. Three groups of ovarian carcinoma orthotopic implantation nude model were applied by PBS (as control) (12 mice), rAAV-Laz (13 mice), rAAV-nm23H(1) (13 mice) intraperitoneally on the day 10 after transplantation. Then the effect of rAAV-nm23H(1) on survival and liver metastatic incident rates in models were observed.rAAV-nm23H(1) and rAAV-Laz were constructed and identified successfully. The titers were both 1 x 10(10) virus particles/ml. The transfection efficiency was 70%. The observation of biological feature showed the orthotopic implantation model was metastatic. The expression of nm23H(1) in AAV-nm23H(1) group was higher significantly than control and rAAV-Laz group (P0.05). Survival time of rAAV-nm23H(1) group was 136.67 days, compared with blank control group 106.67 days and rAAV-Laz group 107.06 days. Kaplan-Meier analysis showed rAAV-nm23H(1) group survived longer than control and rAAV-Laz group (P = 0.0051, P = 0.018 P0.05). The control and rAAV-Laz groups showed no statistically difference in survival time (P = 0.059 P0.05). The metastatic incident rate of control, rAAV-Laz and rAAV-nm23H(1) group was 75%, 61.5%, and 30.8% respectively, the rate of rAAV-nm23H(1) group was lower significantly than that of control and rAAV-Laz group (P = 0.03, P = 0.01). There was no significant difference between control and rAAV-Laz group in liver metastatic rate (P0.05).SW626 line which expressed lower level of nm23H(1) showed high-frequency metastasis properties, rAAV-nm23H(1) could reverse its metastasis in ovarian carcinoma orthotopic transplantation model.

To study the mechanism of multidrug resistance and its reversal by mdr-1 ribozyme in human ovarian cancer.The expression of mdr-1 and p-glycoprotein (p-gp) was studied by confocal laser microscope (Confocal), RT-PCR and Western blot analysis in adriamycin-resistant human ovarian cancer cell line (A2780/ADM) and adriamycin-sensitive one (A2780). The mdr-1 ribozyme was transfected into the A2780/ADM by Lipofectamine 2000 to overcome the multidrug resistance in ovarian cancer.The expression of mdr-1 mRNA and p-gp in A2780/ADM was significantly higher than that in A2780. The expression of mdr-1 mRNA and p-gp in A2780/ADM was lowered after being transfected by mdr-1 ribozyme.Multidrug resistance of A2780/ADM, possibly being caused by overexpression of mdr-1 gene, can be partially reversed by mdr-1 ribozyme.

A new candidate tumor suppressor gene, called phosphatase and tension homology deleted on chromosome ten (PTEN), was the first gene that was found to be phospholipase tumor suppressor gene. Loss of PTEN function by mutation or other mechanisms is closely related to tumorigenesis and progression of multiple carcinomas. This study was designed to investigate the expression of PTEN in carcinogenesis and development of endometrium carcinoma.The expression of PTEN were detected by reverse transcription polymerase chain reaction (RT-PCR) for 24 cases with endometrial carcinoma, 10 cases with endometrial atypical hyperplasia, 10 cases with endometrial hyperplasia,and 10 cases with normal endometrium and by SP immunohistochemical methods for 73 cases with endometrial carcinoma, 25 cases with endometrial atypical hyperplasia, 71 cases with endometrial hyperplasia,and 31 cases with normal endometrium. The results were compared with clinical parameters (histological classification, differentiation, depth of myometrium invasion, and clinical stage).PTEN expression levels of both RNA and protein in patients with endometrial carcinoma and endometrial atypical hyperplasia were significantly lower than those in patients with endometrial hyperplasia and normal endometrium. mRNA relative values were 0.35+/-0.13, 0.46+/-0.11, 2.32+/-0.32, and 2.45+/-0.51, respectively. Loss of PTEN expression rates were 66.67% (38/57), 76.00% (19/25), 5.63% (4/71), and 0 (0/31), respectively. Loss of PTEN expression in patients with endometrial carcinoma was significantly related to histological classification (P0.0001) and differentiation (P=0.0349). It was not related to depth of myometrium invasion and clinical stage(P0.05).Loss of PTEN expression is an early event in endometrial tumorigenesis. Detection of PTEN protein may be a diagnostic biomarker for the earliest endometrial precancers and adenocarcinoma.

Cytotoxic anticancer drugs are less effective in killing tumor cells grown as multicellular spheroids than monolayer cell cultures. The aim of this study was to investigate the molecular mechanism of chemoresistance.Ovarian cancer A2780, CAOV3 multicellular spheroids were obtained from three-dimensional culture. Expression of P-glycoprotein (P-gp) was determined using Western blot analysis and flow cytometry (FCM). The subcellular distribution of P-gp was also determined using laser confocal microscope. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to detect the mdr1 mRNA of both monolayer cells and multicellular spheroids. Cell cycle profiles and apoptosis were also analyzed using FACS. The resistance was detected with trypan blue exclusion testing.Compared with control cells, no expression of P-gp was detected in monolayer cells, but expression of P-gp in aggregate cells was significantly elevated(P0.05). The mdr1 mRNA positive nodes were confirmed by RT-PCR in the aggregate cells. The percentage of cells increased in G(0)-G(1) phase and decreased in S and G(2)-M phase significantly in spheroids cells. Spheroids cells showed higher cell viability than monolayer cells (P(A2780)=0.003, P(CAOV3)=0.015). More apoptotic cells were induced by Taxol in MCS cells than in monolayer cells.Ovarian cancer A2780, CAOV3 multicellular spheroids cultures induced cell resistance to Taxol. High expression of P-gp was induced in ovarian multicellular spheroids and the cells were arrested in G(0)-G(1) phase.

Ataxia telangiectasia is caused by ataxia telangiectasia mutant(ATM) gene and it is characterized by hypersensitivity to the radiation. So the ATM expression should be related to the radiation sensitivity. This study was designed to investigate the relationship between ATM expression in two kinds of tumor cell lines and their cell cycle arrest after irradiation of ionization radiation to explore their self-protection function.ATM mRNA and protein expression of HL-60 and SiHA cell lines were measured by semi-quantitive reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. The cells were irradiated at the dose of 6, 10, and 15 Gy by (60)Co and the change of apoptosis and cell cycle arrest phenomenon at the time of 6, 12, 24, 48, and 60 hours after the radiation were observed.The ATM mean protein fluorescent intensity of HL-60 and SiHA cells were 14.11+/-2.38 and 27.74+/-1.16,respectively. The ATM protein abundance in SiHA was two times more than that in HL-60,and consistent with this, the ATM RNA relative expression of HL-60 and SiHA cells were 0.09 and 0.80, respectively. The ATM transcript levels in SiHA are 9 times more than that in HL-60. The G(2)/M phase arrest after the radiation was observed in both cell lines, whereas SiHA exhibits a much strong cell cycle arrest than HL-60.The cell cycle arrest of HL-60 and SiHA cell lines to the irradiation corresponds to their ATM expression levels. The lower ATM expression, the weaker response for cell cycle arrest induced by radiation.

To block signal transduction of cell cycle checkpoints by antisense blocking of chk1/2 gene to increase the radiation sensitivity of HL-60 cell line.To transfect the HL-60 cell with chk1/2 antisense and sense chain alone and in combination, expose the cells to irradiation at 24 h after the transfection, the chk1 protein change was assayed by Western blot and the cell cycles and annexin V apoptosis rates by FCM.The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line, the apoptotic rate was 26.31% being significantly higher than that of the sense blocking (10.34%) (P0.05), Furthermore, the G(2)/M phase blocking phenomenon decreased and a synergic effect was observed in antisense blocking both the chk1 and chk2 genes.Antisense blocking of chk1/chk2 could increase the apoptotic sensitivity to irradiation.

To explore the role of apoptosis-associated genes and caspase-3 activity in cisplatin (DDP)-resistant human ovarian cancer cell lines.The expressions of apoptosis-associated genes (bcl-2, bax, bcl-X(L) and bcl-X(S)) and the activity of caspase-3 were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot in the cisplatin-resistant (A2780/DDP, COC1/DDP) and sensitive human ovarian cancer cells (A2780 and COC1). The apoptotic rates of A2780, COC1, A2780/DDP and COC1/DDP were measured with flow cytometry when treated with cisplatin.The mRNA expressions of bcl-2 and bcl-X(L) in A2780/DDP cells were 1.87 +/- 0.25 and 1.73 +/- 0.15, and significantly higher than those in A2780 cells (P0.05), which were 1.48 +/- 0.14 and 1.41 +/- 0.19 respectively. The protein expressions of bcl-2 and bcl-X(L) in A2780/DDP cells were 1.99 +/- 0.11 and 1.69 +/- 0.16, and significantly higher than those in A2780 cells (P0.05), which were 1.51 +/- 0.17 and 1.28 +/- 0.11 respectively. The expressions of bcl-2 and bcl-X(L) in COC1/DDP cells were also significantly higher than those in COC1 cells (P0.05). While there was no significant difference in expression of bax between A2780/DDP and A2780 as well as between COC1/DDP and COC1. No expression of bcl-X(S) was detected. When cells treated with different concentration of DDP, the caspase-3 activities, apoptotic rates and PARP expressions in A2780/DDP and COC1/DDP were significantly lower than those in A2780 and COC1 (P0.05), which showed dose-dependent (P0.05).Overexpression of anti-apoptotic genes and decrease of caspase-3 activity may relate to cisplatin resistance in human ovarian cancer cell lines.

To examine the expression of the cyclin-dependent kinase inhibitor p27 in ovarian cancer multicellular spheroid (MCS) and explore the reversal effect of p27-antisense oligodeoxynucleotide (p27-ASON) on taxol resistance in ovarian cancer cell lines.Three-dimensional culture was used to form MCS of human ovarian cancer cell lines A2780 and CAOV3. The MCSs were divided into 3 groups: MCSs transduced with p27-ASON or p27-sense oligodeoxynucleotide (p27-SON) by LipofectAMINE respectively and MCS without transduction. Monolayer A2780 and CAOV3 cells were cultured as controls. The cells were exposed to taxol of different concentrations (0.2, 2.0, 10.0, and 20.0 micro mol/L) for 24 hours. The expression of p27 in those cells was detected with Western blot and flow cytometry (FACS), and the subcellular distribution of p27 was detected by laser confocal microscopy before and after the experiment. The cell cycle profile and apoptosis were analyzed by FACS. The resistance to taxol was detected with trypan blue exclusion testing.Compared with that in monolayer cells, expression of p27 in MCS cells was significantly higher (P(A2780) = 0.011, P(CAOV3) = 0.024). The percentage of cells in G0-G1 phase was significantly higher in the MCS cells than in the monolayer cells, and the percentages of cells in S and G2-M phases were significantly lower in MCS cells than in monolayer cells. The apoptotic rate of the monolayer cells was significantly higher than those of MCS cells treated with taxol of the concentration of 20.0 micro mol/L (P(A2780) = 0.003, P(CAOV3) = 0.015). The apoptotic rate of p27-ASON/MCS was significantly higher than that of MCS (P(A2780) = 0.022, P(CAOV3) = 0.036). There was no significant difference of apoptotic rate between p27-SON/MCS and MCS (P(A2780) = 0.412, P(CAOV3) = 0.071). The monolayer A2780 and CAOV3 cells formed compact spheroids after three-dimensional culture. Cells transduced with p27-SON formed compact MCS too. However, cells tranduced with p27-ASON formed loose collections of cells easy to shatter. The expression of p27 in p27-ASON/MCSs was downregulated.The drug resistance of ovarian cancer MCS is related to upregulation of p27. P27-ASON reverses the resistance of ovarian cancer to taxol, thus increasing the chemotherapeutic sensitivity of ovarian cancer cells.

Cisplatin-based chemotherapy is an important way for treatment of ovarian cancer, but resistance to cisplatin is one of the reasons of treatment failure of ovarian cancer. This study was designed to investigate the relationship between apoptosis-associated proteins and caspase-3 activity as well as their effects on chemoresistance in human ovarian cancer cell lines.The expression of apoptosis-associated proteins (bcl-2, bcl-xL, bax, bcl-xs), the activity of caspase-3 and cleavage of poly ADP-ribose polymerase (PARP) were determined with Western blot analysis in the cisplatin-resistant cell (COC1/DDP) and cisplatin-sensitive human ovarian cancer cell (COC1). The apoptotic ratios of COC1 and COC1/DDP were measured with flow cytometry after treated with different concentration of cisplatin.The expression of bcl-2 and bcl-XL in COC1/DDP cell was significantly higher than that in COC1 cell, whereas the expression of bax showed no change in COC1/DDP and COC1. There was no bcl-xs expression in both COC1 and COC1/DDP cells. The activity of caspase-3, the amount of PARP fragments, and apoptotic ratio in COC1/DDP reduced much more than COC1 did after treated with cisplatin.Cisplatin-resistance in human ovarian cancer cell lines may associated with the overexpression of anti-apoptotic protein bcl-2 and downregulation of caspase-3 activity, but not associated with the expression of bax and bcl-xs.

Tumor orthotopic transplantation model has become the main carrier for tumor animal experiment. However, there was no convenient and reliable method to monitor tumor growth in animal body. This study was designed to evaluate the monitoring impact of urinary beta-human chorionic gonadotropin (beta-HCG) on tumor growth in orthotopic implantation model.Pclone-beta-HCG stably transfected human ovarian carcinoma cell line A2780-CG was made as the orthotopic implantation model. The urinary beta-HCG/creatine ratio was continuously determined and plotted into curve, so as to indirectly know the rule of tumor growth in nude mice and the monitoring value of the ratio for intraperitoneal chemotherapy by cisplatin.The tumorigenesis time in beta-HCG system transfected tumor cell A2780-CG was similar to that in the original cell line. The content of urinary beta-HCG/creatine had a positive correlation to tumor weight in animal body (r = 0.98); After chemotherapy with cisplatin, the beta-HCG/creatine ratio showed progressively decreased.beta-HCG system could be used to sensitively and non-invasively monitor the tumor growth in orthotopic implantation model of human ovarian carcinoma in nude mice. Meanwhile, it provides a new convenient monitoring method for tumor treatment in the model.