Peroxiredoxin I and II as novel therapeutic molecular targets in cervical cancer treatment through regulation of endoplasmic reticulum stress induced by bleomycin.

Cervical cancer, significantly affecting women worldwide, often involves treatment with bleomycin, an anticancer agent targeting breast, ovarian, and cervical cancers by generating reactive oxygen species (ROS) to induce cancer cell death. The Peroxiredoxin (PRDX) family, particularly PRDX1 and 2, plays a vital role in maintaining cellular balance by scavenging ROS, thus mitigating the damaging effects of bleomycin-induced mitochondrial and cellular oxidative stress. This process reduces endoplasmic reticulum (ER) stress and prevents cell apoptosis. However, reducing PRDX1 and 2 levels reverses their protective effect, increasing apoptosis. This research highlights the importance of PRDX1 and 2 in cervical cancer treatments with bleomycin, showing their potential to enhance treatment efficacy by managing ROS and ER stress and suggesting a therapeutic strategy for improving outcomes in cervical cancer treatment.

Hispidin Increases Cell Apoptosis and Ferroptosis in Prostate Cancer Cells Through Phosphatidylinositol-3-Kinase and Mitogen-activated Protein Kinase Signaling Pathway.

BACKGROUND/AIM: Chemotherapy is mainly used in the clinical treatment of prostate cancer. Different anticancer mechanisms can induce cell death in various cancers. Reactive oxygen species (ROS) play crucial roles in cell proliferation, differentiation, apoptosis, and signal transduction. It is widely accepted that ROS accumulation is closely related to chemical drug-induced cancer cell death. MATERIALS AND METHODS: We utilized the MTT assay to detect changes in cell proliferation. Additionally, colony formation and wound healing assay were conducted to investigate the effect of hispidin on cell colony formation and migration ability. Fluorescence microscopy was used to detect intracellular and mitochondrial ROS levels, while western blot was used for detection of cell apoptosis. RESULTS: Hispidin treatment significantly decreased viability of PC3 and DU145 cancer cells but exhibited no cytotoxicity in WPMY-1 cells. Furthermore, hispidin treatment inhibited cell migration and colony formation and triggered cellular and mitochondrial ROS accumulation, leading to mitochondrial dysfunction and mitochondrion-dependent apoptosis. Moreover, hispidin treatment induced ferroptosis in PC3 cells. Scavenging of ROS with N-acetyl cysteine significantly inhibited hispidin-induced apoptosis by altering the expression of apoptosis-related proteins, such as cleaved caspase-3, 9, Bax, and Bcl2. Furthermore, hispidin treatment dramatically up-regulated MAPK (involving p38, ERK, and JNK proteins) and NF-kB signaling pathways while down-regulating AKT phosphorylation. Hispidin treatment also inhibited ferroptosis signaling pathways (involving P53, Nrf-2, and HO-1 proteins) in PC3 cells. In addition, inhibiting these signaling pathways via treatment with specific inhibitors significantly reversed hispidin-induced apoptosis, cellular ROS levels, mitochondrial dysfunction, and ferroptosis. CONCLUSION: Hispidin may represent a potential candidate for treating prostate cancer.

PRDX1 negatively regulates bleomycin-induced pulmonary fibrosis via inhibiting the epithelial-mesenchymal transition and lung fibroblast proliferation in vitro and in vivo.

BACKGROUND: Pulmonary fibrosis is a major category of end-stage changes in lung diseases, characterized by lung epithelial cell damage, proliferation of fibroblasts, and accumulation of extracellular matrix. Peroxiredoxin 1 (PRDX1), a member of the peroxiredoxin protein family, participates in the regulation of the levels of reactive oxygen species in cells and various other physiological activities, as well as the occurrence and development of diseases by functioning as a chaperonin. METHODS: Experimental methods including MTT assay, morphological observation of fibrosis, wound healing assay, fluorescence microscopy, flow cytometry, ELISA, western blot, transcriptome sequencing, and histopathological analysis were used in this study. RESULTS: PRDX1 knockdown increased ROS levels in lung epithelial cells and promoted epithelial-mesenchymal transition (EMT) through the PI3K/Akt and JNK/Smad signalling pathways. PRDX1 knockout significantly increased TGF-ß secretion, ROS production, and cell migration in primary lung fibroblasts. PRDX1 deficiency also increased cell proliferation, cell cycle circulation, and fibrosis progression through the PI3K/Akt and JNK/Smad signalling pathways. BLM treatment induced more severe pulmonary fibrosis in PRDX1-knockout mice, mainly through the PI3K/Akt and JNK/Smad signalling pathways. CONCLUSIONS: Our findings strongly suggest that PRDX1 is a key molecule in BLM-induced lung fibrosis progression and acts through modulating EMT and lung fibroblast proliferation; therefore, it may be a therapeutic target for the treatment of BLM-induced lung fibrosis.

GRADE Use in Evidence Syntheses Published in High-Impact-Factor Gynecology and Obstetrics Journals: A Methodological Survey.

Objective: To identify and describe the certainty of evidence of gynecology and obstetrics systematic reviews (SRs) using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach. Method: Database searches of SRs using GRADE, published between 1 January 2016 to 31 December 2020, in the 10 "gynecology and obstetrics" journals with the highest impact factor, according to the Journal Citation Report 2019. Selected studies included those SRs using the GRADE approach, used to determine the certainty of evidence. Results: Out of 952 SRs, ninety-six SRs of randomized control trials (RCTs) and/or nonrandomized studies (NRSs) used GRADE. Sixty-seven SRs (7.04%) rated the certainty of evidence for specific outcomes. In total, we identified 946 certainty of evidence outcome ratings (n = 614 RCT ratings), ranging from very-low (42.28%) to low (28.44%), moderate (17.65%), and high (11.63%). High and very low certainty of evidence ratings accounted for 2.16% and 71.60% in the SRs of NRSs, respectively, compared with 16.78% and 26.55% in the SRs of RCTs. In the SRs of RCTs and NRSs, certainty of evidence was mainly downgraded due to imprecision and bias risks. Conclusions: More attention needs to be paid to strengthening GRADE acceptance and building knowledge of GRADE methods in gynecology and obstetrics evidence synthesis.

Phytosterol intake and overall survival in newly diagnosed ovarian cancer patients: An ambispective cohort study.

Background: Phytosterol is a bioactive compound existing in all plant foods, which might have anticancer properties. The aim of this study was to first assess the impact of the pre-diagnosis phytosterol intake on overall survival (OS) of patients with ovarian cancer (OC). Materials and methods: This ambispective cohort study recruited 703 newly diagnosed OC patients to investigate the aforementioned associations. Dietary intake was assessed using a validated 111-item food frequency questionnaire. Deaths were ascertained until March 31, 2021, through active follow-up and medical records. Cox proportional hazards regression models were applied to calculate the hazard ratios (HRs) and 95% confidence intervals (CIs). Results: During the median follow-up of 37.17 months, 130 deaths occurred. The median age at diagnosis of 703 OC patients was 53.00 (interquartile: 48.00-60.00) years. Of these, almost half patients (48.08%) were diagnosed in advanced International Federation of Gynecology and Obstetrics (FIGO) stage (III-IV). Additionally, more than half patients were serous carcinoma (68.14%), poorly differentiated (85.21%), and no residual lesions (78.66%). Patients consumed the highest tertile of dietary campesterol (HR = 0.54, 95% CI = 0.31-0.94, P trend < 0.05), stigmasterol (HR = 0.60, 95% CI = 0.37-0.98), and ß-sitosterol (HR = 0.63, 95% CI = 0.40-0.99) were significantly associated with better OS compared with those with the lowest tertile of intake. The curvilinear associations were observed between total phytosterols and ß-sitosterol intake and OC survival (P non-linear < 0.05). Significant associations were generally consistent across different subgroups stratified by demographical, clinical, and immunohistochemical characteristics. Moreover, there were significant interactions between phytosterol intake and age at diagnosis, body mass index, as well as expressions of Wilms' tumor-1 and Progestogen Receptor (all P interaction < 0.05). Conclusion: Pre-diagnosis higher campesterol, stigmasterol, and ß-sitosterol intake were associated with better survival among OC patients.

Pre-diagnosis Cruciferous Vegetables and Isothiocyanates Intake and Ovarian Cancer Survival: A Prospective Cohort Study.

Background: The associations of the consumption of cruciferous vegetables (CVs) and their bioactive components, isothiocyanates (ITCs), with ovarian cancer (OC) mortality have been unclear, owing to limited studies and inconsistent findings. To date, no studies have evaluated these associations among Chinese patients with OC. This study aims to provide more evidence indicating the relationships of pre-diagnosis CVs and ITC intake with OC survival. Methods: We examined the associations of pre-diagnosis CV and ITC intake with OC mortality in a hospital-based cohort (n = 853) of Chinese patients with epithelial OC between 2015 and 2020. Pre-diagnosis dietary information was evaluated with a validated food frequency questionnaire. Deaths were ascertained until March 31, 2021 via medical records and active follow-up. The associations were examined with the Cox proportional hazards model, adjusted for potential confounders, and stratified by menopausal status, residual lesions, histological type, and body mass index (BMI). Results: During a median follow-up of 37.2 months (interquartile: 24.7-50.2 months), we observed 130 deaths. The highest tertile of total CV intake was associated with better survival than the lowest tertile intake [hazard ratio (HR) = 0.57, 95% confidence interval (CI) = 0.33-0.98, p trend < 0.05]. In addition, higher intake of ITCs from CVs was associated with better survival (HRT3VS.T1 = 0.59, 95% CI = 0.36-0.99, p trend = 0.06). Significant inverse associations were also observed for subgroup analyses stratified by menopausal status, residual lesions, histological type, and BMI, although not all associations showed statistical significance. Conclusion: Increasing pre-diagnosis consumption of CVs and ITCs was strongly associated with better survival in patients with OC.

Human epididymis protein 4 promotes P­glycoprotein­mediated chemoresistance in ovarian cancer cells through interactions with Annexin II.

The aim of the present study was to investigate the effects of human epididymis protein 4 (HE4) on drug resistance and its underlying mechanisms. The associations among proteins were detected by immunoprecipitation and immunofluorescence assays. Then, stably transfected cell lines CAOV3­HE4­L and CAOV3­A2­L expressing HE4 short hairpin (sh)RNAs and ANXA2 shRNAs, respectively, were constructed. MTT assay, immunocytochemistry, western blotting, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and flow cytometry were employed to examine drug sensitivity, as well as the expression and activity of P­glycoprotein (P­gp). HE4 and P­gp in epithelial ovarian cancer tissues were assessed via immunohistochemistry. MicroRNAs that targeted the P­gp gene, ABCB1, were predicted using bioinformatics methods, and their expression was evaluated by RT­qPCR. The common signaling pathways shared by HE4, ANXA2 and P­gp were selected by Gene Set Enrichment Analysis (GSEA). The interaction of HE4, ANXA2 and P­gp were confirmed. P­gp expression was positively associated with HE4 and ANXA2 expression, respectively. Moreover, it was observed that there was no significant rescue of P­gp expression in CAOV3­A2­L cells following the administration of active HE4 protein. In addition, the expression of HE4 and P­gp in ovarian cancer tissues of drug­resistant patients were higher compared with that of the drug­sensitive group (P<0.05). Furthermore, the results revealed that hsa­miR­129­5p was significantly increased accompanied by decreased HE4 or ANXA2 expression and P­gp expression in CAOV3­HE4­L and CAOV3­A2­L cells. GSEA analyses disclosed that HE4, ANXA2 and P­gp genes were commonly enriched in the signaling pathway involved in regulating the actin cytoskeleton. These results indicated that HE4 promotes P­gp­mediated drug resistance in ovarian cancer cells through the interactions with ANXA2, and the underlying mechanism may be associated with decreased expression of hsa­miR­129­5p and dysregulation of the actin cytoskeleton signaling pathway.

The compound 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione leads to apoptotic cell death by increasing the cellular reactive oxygen species levels in Ras-mutated liver cancer cells.

The aim of the present study was to verify the pro-apoptotic anticancer potential of several 5,8-dimethoxy-1,4-phthoquinone (DMNQ) derivatives in Ras-mediated tumorigenesis. MTT assays were used to detect cellular viability and flow cytometry was performed to assess intracellular reactive oxygen species (ROS) levels and apoptosis. The expression levels of proteins were detected via western blotting. Among the 12 newly synthesized DMNQ derivatives, 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione (BZNQ; component #1) significantly reduced cell viability both in mouse NIH3T3 embryonic fibroblasts cells (NC) and H-RasG12V transfected mouse NIH3T3 embryonic fibroblasts cells (NR). Moreover, BZNQ resulted in increased cytotoxic sensitivity in Ras-mutant transfected cells. Furthermore, the reactive oxygen species (ROS) levels in H-RasG12V transfected HepG2 liver cancer cells (HR) were significantly higher compared with the levels in HepG2 liver cancer cells (HC) following BZNQ treatment, which further resulted in increased cellular apoptosis. Eliminating cellular ROS using an ROS scavenger N-acetyl-L-cysteine markedly reversed BZNQ-induced cellular ROS accumulation and cell apoptosis in HC and HR cells. Western blotting results revealed that BZNQ significantly downregulated H-Ras protein expression and inhibited the Ras-mediated downstream signaling pathways such as protein kinase B, extracellular signal-related kinase and glycogen synthase kinase phosphorylation and ß-catenin protein expression. These results indicated that the novel DMNQ derivative BZNQ may be a therapeutic drug for Ras-mediated liver tumorigenesis. The results of the current study suggest that BZNQ exerts its effect by downregulating H-Ras protein expression and Ras-mediated signaling pathways.

Systematic profiling of alternative splicing signature reveals prognostic predictor for cervical cancer.

AIM: Cervical cancer is a common malignant carcinoma of the gynecological tract with high morbidity and mortality. Therefore, it is crucial to elucidate the pathogenesis, prevention, diagnosis and prognosis of cervical cancer by searching for the involved key genes. METHOD: In this study, the alternative splicing (AS) events of 253 patients with cervical cancer were analyzed, and 41,766 AS events were detected in 9961 genes. Univariate analysis was performed to screen prognostic AS events. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to identify the pathways in which these AS events were involved. RESULTS: We found that exon skip (ES) is the main AS event in patients with cervical cancer. There was pronounced consistency between the genes involved in overall survival and those involved in recurrence. At the same time, we found that a gene may exhibit several different types of AS events, and these different AS events may be related to prognosis. Four characteristic genes, HSPA14, SDHAF2, CAMKK2 and TM9SF1, that can be used as prognostic markers for cervical cancer were selected. CONCLUSION: The importance of AS events in the development of cervical cancer and prediction of prognosis was revealed by a large amount of data at the whole genome level, which may provide a potential target for cervical cancer treatment. We also provide a new method for exploring the pathogenesis of cervical cancer to determine clinical treatment and prognosis more accurately.

Expressions of CD44,CD47,and c-met in Ovarian Clear Cell Carcinoma and Their Clinical Significance.

Objective To investigate the expressions of CD44,CD47,and c-met in ovarian clear cell carcinoma (OCCC) tissue and their correlations with clinical variables and prognosis. Methods Immunohistochemical method was used to investigate the expressions of CD44,CD47,and c-met in tissues from 86 OCCC patients and the relationships of their expressions with the clinicopathological factors of OCCC were analyzed. Results The expressions of CD44,CD47,and c-met were significantly high in OCCC tissues (90.7%,91.9%,and 94.2%,respectively). The strong positive expressions of CD44 and CD47 were significantly correlated with advanced International Federation of Gynecology and Obstetrics stages,chemotherapeutic resistance,and poor prognosis (all P<0.05),the strong positive expression of c-met was significantly correlated with chemotherapeutic resistance and poor prognosis (all P<0.05),whereas there was no correlation between the strong positive expressions of CD44,CD47,and c-met and the lymphatic node metastasis. COX survival analysis revealed that advanced International Federation of Gynecology and Obstetrics stages and high expressions of CD44,CD47 and c-met were independent risk factors for poor prognosis (P<0.05). There was a positive correlation between CD44 (or CD47) and c-met and between CD44 and CD47 (the Spearman correlation coefficient rs was 0.783,0.776,and 0.835,respectively,all P<0.01). Conclusions The expressions of CD44,CD47,and c-met increase in OCCC tissues and are correlated with each other. High expressions of CD44,CD47,and c-met are independent factors for poor prognosis.

Lewis Y regulates signaling molecules of the transforming growth factor ß pathway in ovarian carcinoma-derived RMG-I cells.

LeY (Lewis Y) is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of LeY is frequently observed in epithelial-derived cancers and is correlated to pathological staging and prognosis. To study the role of LeY on cancer cells, a stably LeY-overexpressing cell line, RMG-I-H, was developed previously by transfection of the α1,2-fucosyltransferase gene, a key enzyme that catalyzes the synthesis of LeY, into ovarian carcinoma-derived RMG-I cells. Our studies have shown that LeY is involved in the changes in biological behavior of RMG-I-H cells. However, the mechanism is still largely unknown. In this study, we determined the structural relationship and co-localization between LeY and TßRI/TßRII, respectively, and the potential cellular signaling mechanism was also investigated. We found that both TßRI and TßRII contain the LeY structure, and the level of LeY in TßRI and TßRII in RMG-I-H cells was significantly increased. Overexpression of LeY up-regulates the phosphorylation of ERK, Akt and down-regulates the phosphorylation of Smad2/3. In addition, the phosphorylation intensity was attenuated significantly by LeY monoantibody. These findings suggest that LeY is involved in the changes in biological behavior through TGF­ß receptors via Smad, ERK/MAPK and PI3K/Akt signaling pathways. We suggest that LeY may be an important composition of growth factor receptors and could be an attractive candidate for cancer diagnosis and treatment.

Expression and correlation of Lewis y antigen and TGF-ß1 in ovarian epithelial carcinoma.

Lewis y is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Elevation of Lewis y is frequently observed in epithelial-derived cancers. This study aimed to detect the expression and clinical significance of the Lewis y antigen and TGF-ß1 (transforming growth factor ß1) in ovarian epithelial tumors, and to evaluate the correlation between them. Immunohistochemical staining was used to detect the expression of Lewis y antigen and TGF-ß1 in 60 cases of ovarian epithelial malignant tumors, 20 cases of borderline ovary tumors, 20 cases of benign ovary tumors and 10 cases of normal ovarian tissues. An immunofluorescence double labeling method was also used to detect the correlation between Lewis y antigen and TGF-ß1. The positive rates of Lewis y antigen in ovarian epithelial cancer tissues was 88.33%, significantly higher compared to those of borderline ovarian tumors (60.00%) (P<0.05), benign ovarian tumors (35.00%) (P<0.01) and normal ovarian tissues (0%) (P<0.01). Its expression was not associated with clinical parameters; the positive rates of TGF-ß1 in ovarian epithelial cancers were 78.33%, significantly higher compared to those of benign ovarian tumors (65.00%) (P<0.05) and normal ovarian tissues (40.00%) (P<0.05); the positive rates of the TGF-ß1 and Lewis y were not associated with metastasis of lymph nodes and histological types, differentiation degree and clinical stage (P>0.05). Expression of Lewis y antigen and TGF-ß1 was significantly positively associated with epithelial carcinoma. Close correlation between Lewis y, TGF-ß1 and ovarian cancer was observed. Altered expression of Lewis y antigen may cause changes in TGF-ß1 expression. Lewis y can increase the growth of ovarian cancer cells and the invasion ability by promoting TGF-ß1 abnormal expression and by promoting angiogenesis and a change in its signal transduction pathway. This study provides theoretical evidence for the development of ovarian cancer biological treatments.

[Relationship between PD-1 expression on peripheral T lymphcytes and HBeAg seroconversion after entecavir treatment in chronic hepatitis B patients].

To observe longitudinally the expression of Programmed death 1(PD-1) on peripheral blood T cells in chronic hepatitis B patients underwent antiviral treatment with entecavir (ETV) and to explore the relationship between PD-1 expression and HBeAg seroconversion. Twenty HBeAg positive patients underwent antiviral treatment with ETV were followed up for 51 weeks. 14 patients remained HBeAg positive and 6 patients achieved HBeAg seroconversion. Peripheral blood was collected at six time points: T0: baseline, T1: 2-4week; T2: 5-10week; T3: 11-20week; T4: 21-30week: T5: 31-51week. PD-1 expressions on T cells were assessed by flow cytometry. Serum HBV DNA loads were determined by real-time fluorescent quantitative polymerase chain reaction (PCR) and serum ALT levels were examined at the same time. At baseline, serum HBV DNA load of patients without HBeAg seroconversion and with HBeAg seroconversion were (7.54+/-0.67) log10 copies/ml and (7.30+/-0.79) log10 copies/ml (P more than 0.05), the ALT levels were (187.26+/-184.15) U/L and (272.17+/-215.07) U/L (P more than 0.05), PD-1 exprissions on CD4+ T cells were 6.04%+/-3.71% and 6.77%+/-2.88% (P more than 0.05), PD-1 exprissions on CD8+ T cells were 6.39%+/-3.33% and 8.88%+/-2.84% (P more than 0.05). After ETV treatment, serum HBV DNA loads and ALT levels both decreased gradually, which was positively correlated with PD-1 expressions on CD4+ and CD8+ T cells (r=0.212, P = 0.05; r=0.377, P less than 0.01; r=0.279, P less than 0.05; r=0.347, P less than 0.01). During the same monitoring period, the HBV DNA loads, ALT levels and PD-1 expressions on T cells of the patients with HBeAg seroconversion decreased significantly as compared with the patients without HBeAg seroconversion. Besides, the decrease of HBV DNA loads during period deltaT0-T1 and deltaT0-T2 and PD-1 expressions on CD8+ T cells during period deltaT0-T2 and deltaT0-T3 were significantly different between these two kinds of patients (49.9% vs 37.3%, P less than 0.05; 56.7% vs 47.4%, P less than 0.05; 70.1% vs -4.2%, P less than 0.05; 66.9% vs 24.5%, P less than 0.05). The rapid decrease of PD-1 expression on peripheral CD8+ T cells after antiviral treatment with ETV is positvely correlated with the decrease of serum HBV DNA loads and may be used as a predictive index for HBeAg seroconversion in HBeAg positive patients.

Development of the personalized criteria for microscopic review following four different series of hematology analyzer in a Chinese large scale hospital.

BACKGROUND: A generally accepted guideline ("41 rules") published by the International Consensus Group for Hematology Review (ICGHR) can not be suitable for all the laboratories because the facility type, laboratory requirements, sample volume, review rate, turn around time, instrument model and characters etc. are quite different from each other, which may cause a higher workload for microscopy review or lead to false or misleading results. Therefore, we decided to develop the personalized review criteria for 4 series of hematology analyzers in the same hospital, and describe all the implement procedures in detail. METHODS: The total 1770 blood samples were collected from Peking Union Medical College Hospital. Referring to the suggested criteria by international consensus group for hematology review ("41 rules"), the personalized review criteria for 4 series of hematology analyzers including Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i were established and validated by adjusting the rules in order to reduce the false positive rate and keep the false negative acceptable by clinical. RESULTS: Using the "41 rules", high review rates of 37.94%, 35.56%, 33.44% and 37.94% were got respectively in Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i. Three false positive rules mainly were observed in all of 4 analyzers: white blood cell < 3 × 10(9)/L or >30 × 10(9)/L, platelet < 100 × 10(9)/L or > 1000 × 10(9)/L and immature granulocyte. Specialized rules were observed in different series of analyzers, atypical/variant lymphs flag were found mainly in Sysmex XE-2100, Aniso-RBC were found mainly in Sysmex XT-1800i, flag of "immature granulocyte" mainly in Sysmex XS-800i, Micro-RBC, Macro-RBC and Aniso-RBC mainly in Siemens Advia 2120. Rules of immature granulocyte, blast, and NRBC flag would be mainly triggered by hematology malignant tumor. We could not delete these rules due to the risk of false negative of serious disease, other rules were deleted or revised. After continually optimizing to the rules, we finalized the criteria suitable for Siemens Advia 2120, Sysmex XE-2100, Sysmex XT-1800i and Sysmex XS-800i in our laboratory. The false negative rates were 2.94%, 2.86%, 3.10% and 2.78%, the review rates were 31.07%, 30.00%, 30.01% and 30.09%, and there was no hematology malignant tumor missed. Validated by 547 samples, the false negative rates of our optimized rules were 0.37%, 0.55%, 0.55%, and 0.91% respectively. CONCLUSION: The criteria can be based on the criteria established by International Consensus Group for Hematology Review but must be optimized according to the different requirements.

Lewis(y) antigen stimulates the growth of ovarian cancer cells via regulation of the epidermal growth factor receptor pathway.

Lewis(y) antigen is an oligosaccharide containing two fucoses, and is expressed variously in 75% of ovarian tumors, where its high expression level predicts poor prognosis. The effect and the possible mechanism of Lewis(y) on the proliferation of human ovarian cancer cells are still largely unkown. We report here that transfecting alpha1,2-FT gene into RMG-I cells increased the expression of Lewis(y) and promoted cell proliferation. In alpha1,2-FT-transfected cells, the Lewis(y) content of EGFR was increased dramatically. Tyrosine phosphorylation of EGFR was elevated. Concomitantly, tyrosine phosphorylation of Akt, ERK1/2 was also upregulated. Moreover, the expression of HER2/neu mRNA and protein, the tyrosine phosphorylation of HER2/ neu were also elveated, while the expression of p27 was significantly reduced. However, the expression of EGFR and the relative content of Lewis(y) on HER2/neu were unchanged. The above-mentioned alterations were correlated with the Lewis(y) content of EGFR and alpha1,2-FT expression in cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different expression of alpha1,2-FT were attenuated significantly by the inhibitor of EGFR tyrosine kinase and by the mono-antibody to Lewis(y). Meanwhile, the reduction in p27 and the difference in its expression among the two cell lines were also blocked by the Lewis(y) antibody. The PI3K signaling pathway was more important than the MAPK pathway in the regulation of p27 expression. These findings provide strong evidence that increased expression of Lewis(y) promotes cell proliferation through regulating the phosphorylation and expression of some molecules involved in the EGFR/PI3K-signaling pathway.

Enhancement of the adhesive and spreading potentials of ovarian carcinoma RMG-1 cells due to increased expression of integrin alpha5beta1 with the Lewis Y-structure on transfection of the alpha1,2-fucosyltransferase gene.

Le(Y) antigen is known to be associated with malignant properties including metastasis and a poor prognosis of ovarian carcinomas. To clarify the mechanisms underling these properties, we established ovarian carcinoma-derived cells exhibiting enhanced expression of Le(Y) by transfection with alpha1,2-fucosyltransferase and compared their cellular properties with those of the original cells. So the human alpha1,2-fucosyltransferase gene was transfected into ovarian carcinoma-derived RMG-1 cells, which are known to contain Le(X), a precursor of Le(Y), and RMG-1-hFUT cells exhibiting enhanced expression of Le(Y) were established by selection with anti-Le(Y) antibodies, and their adhesive and spreading potentials on fibronectin-coated plates were compared with those of RMG-1 cells. Results showed that the relative expression of Le(Y) in RMG-1-hFUT cells was about 20-fold that in RMG-1 cells, and that of integrin alpha5beta1 and an integrin-mediated signal transduction molecule, focal adhesion kinase, was also increased in RMG-1-hFUT cells. Interestingly, anti-Le(Y) antibodies were revealed to immunoprecipitate integrin alpha5beta1, indicating that its oligosaccharides are composed of Le(Y), the amounts of which was substantially elevated in RMG-1-hFUT cells. The adhesion and spreading potentials on fibronectin-coated plates of RMG-1-hFUT cells were significantly enhanced in comparison to those of RMG-1 cells, and were greatly suppressed by anti-Le(Y) antibodies, indicating that Le(Y) is involved in the integrin-fibronectin interaction. These results suggested that transfection of the alpha1,2-fucosyltransferase gene into ovarian carcinoma-derived cells brought about elevated expression of integrin alpha5beta1 with Le(Y), resulting in enhancement of the adhesion and spreading potentials of cells through the integrin-fibronection interaction, which was inhibited by anti-Le(Y) antibodies. Thus, Le(Y) in integrin alpha5beta1 was thought to be involved in the enhanced cell adhesion properties of malignant ovarian carcinomas.

[Effect of Lewis y antigen on regulating gene expression of partial drug resistance associated proteins in human ovarian cancer cell line RMG-I-H].

OBJECTIVE: To investigate the influence of Lewis y antigen on the gene expression of partial drug resistance associated proteins in human ovarian cancer cell line RMG-I-H. METHODS: RT-PCR was used to determine the gene expressions of partial drug resistance associated proteins in RMG-I-H cell line transfected with alpha1, 2-fucosyltransferases gene and RMG-I cell line, as well as in RMG-I-H treated with or without anti-Lewis y monoclonal antibody at the concentration of 10 micro/g/ml. The immunocytochemical method was used to detect the expression of P-glycoprotein (P-gp) in RMG-I and RMG-I-H cell lines. RMG-I and RMG-I-H cells were transplanted into nude mice and the expression of P-gp in the tissues was measured by immunohistochemistry. RESULTS: The mRNA expressions of protein kinase C-alpha (PKC-alpha), topoismerase I ( Topo I ), multidrug resistance-associated protein-1 (MRP-1), and MRP-2 were significantly higher in RMG-I-H cells than those in RMG-I cells (0.46 +/- 0.02 vs. 0.27 +/- 0.05, 0.82 +/- 0.08 vs. 0.52 +/- 0.04, 0.66 +/- 0.07 vs. 0.34 +/- 0.12, and 0.44 +/- 0.08 vs. 0.23 +/- 0.05; all P < 0.05). However, the mRNA expression of multi-drug resistance 1 (MDR-1) was significantly lower in RMG-I-H cells than that in RMG-I cells (0.26 +/- 0.05 vs. 0.45 +/- 0.08, P < 0.05). The P-gp level increased in RMG-I-H cells compared with that in RMG-I cells both in vivo and in vitro (P < 0.05). Expressions of MDR-1, MRP-1, MRP-2, PKC-alpha, and Topo I mRNA decreased by the time in RMG-I-H cells treated with anti-Lewis y monoclonal antibody (all P < 0.05), while mRNA expressions of those genes in the control group did not statistically change (P > 0.05). In addition, MDR-1, MRP-1, MRP-2, PKC-alpha, and Topo I mRNA expressions were significantly lower in RMG-I-H cells treated with anti-Lewis y monoclonal antibody than those in the control group at 6 hours (all P < 0.05) and the inhibition ratios were 48.55%, 77.50%, 70.18%, 45.86%, and 46.13%, respectively. CONCLUSION: The Lewis y antigen of the human ovarian cancer cell surface is closely correlated with the regulation on the gene expression of partial drug resistance associated proteins.

[Impact of alpha1,2-fucosyl transferase gene transfection on cancer-related gene expression profile of human ovarian cancer cell line RMG-1].

BACKGROUND & OBJECTIVE: The malignant biological behaviors are enhanced in human ovarian cancer cell line RMG-1 after transfection of alpha1,2-fucosyl transferase (alpha1,2-FT) gene. This study was to investigate the influence of alpha1,2-FT gene transfection on cancer-related gene expression profile of RMG-1 cells. METHODS: Gene expressing vector pcDNA3.1-HFT-H and empty vector pcDNA3.1 were transfected into RMG-1 cells to produce RMG-1-H and RMG-1-C cells separately. Gene expression profiles of these two cell lines were detected by gene chip assay. The acquired data were inquired on the GoMiner online database. RESULTS: After transfection, comparing with those in RMG-1-C cells, 88 differentially expressed genes were identified in RMG-1-H cells: 60 were up-regulated and 28 were down-regulated. These genes are involved in protein binding, nucleotide binding, cell proliferation, DNA-dependent regulation of transcription, signal transduction, protein amino acid phosphorylation, transcription, cell adhesion, and so on. CONCLUSION: The transfection of alpha1,2-FT gene causes the changes of gene expression profile in ovarian cancer RMG-1 cells.

[Transfection of alpha1, 2-fucosyltransferase gene increases the antigenic expression of Lewis y in ovarian cancer cell line RMG-I].

OBJECTIVE: To transfect human alpha1, 2-fucosyltransferase (alpha1, 2-FT) gene to ovarian cancer cell line RMG-I and investigate the antigenic expression change of Lewis y and the other related oligosaccharides. METHODS: The expression vector pcDNA3.1(-)-HFUT-H was constructed by polymerase chain reaction (PCR) to clone human alpha1, 2-FT gene coding region. The alpha1, 2-FT gene stable high-expression cell line RMG-I-H was established by transfecting pcDNA3.1(-)-HFUT-H to ovarian cell line RMG-I. The change of alpha1, 2-FT activity in the cell line before and after the tranfection was confirmed by the determination of enzymatic activity. The changes of cell lipid and glucolipid, especially the change of type II oligosaccharide, in the cell line before and after the transfection was determined by Thin-Layer Chromatography (TLC) and TLC immunostaining method, respectively. RESULTS: The H-1 antigen and Lewis y antigen were obviously increased in the cell line RMG-1-H, especially the latter one, which was 20 times higher than before, and the type I saccharide chain Lewis b was decreased significantly. The main lipid components on the cell membrane, cholesterol and phosphatides, showed no change in the cell lines before and after the transfection, and the neutral glycolipid also showed no obvious change. CONCLUSIONS: The transfection of alpha1, 2-FT gene can increase the activity of alpha1, 2-FT in the cell line RMG-I and mainly increase the expression of Lewis y antigen simultaneously. The construction of RMG-I Lewis y high expression cell line provides a cell model for further study on the relationship between Lewis y antigen and biological behaviors in the ovarian cancer.

[Inhibitory effect of anti-Lewis y antibody on alpha1,2-fucosyltransferase gene transfected human ovarian cancer cells in vitro].

AIM: To study the inhibitory effect of anti-Lewis y monoclonal antibody on proliferation, adhesion and invasion of alpha1, 2-fucosyltransferase gene transfected human ovarian cancer cell line RMG-I-H with over-expression of Lewis y antigen in vitro. METHODS: The changes of proliferation, adhesion and invasion of RMG-I-H cells in vitro were detected by cell growth assay, cell adhesive assay and cell invasive assay, respectively, with goat anti-human IgG antibody treated group or non-antibody treated group as control. RESULTS: The proliferation and adhesion of RMG-I-H cells were remarkably inhibited by preincubation of the tumor cells with anti-Lewis y antibody in vitro (P<0.05). The growth inhibitory rate from the second day to the seventh day was 5.62%, 19.75%, 34.96%, 46.51%, 49.78% and 33.33%, respectively. The adhesion inhibitory rate at different time points (15, 30, 60 min) was 47.59%, 58.64% and 13.85%, respectively. Invasive assay with a transwell cell culture chamber showed that there were no significant differences of invasion in vitro between each concentration of the Lewis y antibody treated group and non-antibody treated group (P>0.05). CONCLUSION: Anti-Lewis y antibody significantly inhibits the proliferation and adhesion of RMG-I-H cells cultured in vitro, but it has no effect on invasion, which indicates that Lewis y is associated with some biological behaviors, such as proliferation and adhesion. The antibody against Lewis y may be an effective anti-metastasis agent for the treatment of patients with ovarian cancer.