Changes in the fucose content of haptoglobin in breast and ovarian cancer: Association with disease progression

There is increasing evidence for changes in fucosylation in cancer. Previously, we showed that the fucose-specific lectin,Lotus tetragonolobus, extracts an abnormal form of haptoglobin (Hp) from cancer sera. This study investigates the monosaccharide content of Hp obtained from women with ovarian and breast cancer at different stages of their disease. In both cancers, Hp fucose was low when the disease was benign or in remission and much higher when the disease was progressive. This occurred whether the data was expressed per mole of protein or per three mannose residues. Changes in other monosaccharides were minor compared with fucose. There were small increases in theN-acetylglucosamine and galactose content (per three mannoses) in ovarian cancer, suggesting that some glycan chains have increased branching. The latter was independent of disease activity which may be due to some indirect cause such as cytotoxic therapy or an inflammatory response. When ovarian cancer patients were in remission, the number of glycosylation sites on Hp was reduced. Hp isolated from patients with early, but not advanced breast cancer also appeared to have increased glycan branching. The increased fucosylated Hp may interfere with fucose-mediated adhesion reactions of cancer cells.Part of this work was published in abstract form,Glycoconjugate J 1993;10; 318.     

Nitidine chloride inhibited the expression of S phase kinase-associated protein 2 in ovarian cancer cells

Nitidine chloride (NC) has been reported to exert its anti-tumor activity in various types of human cancers. However, the molecular mechanism of NC-mediated tumor suppressive function is largely unclear. In the current study, we used several approaches such as MTT, FACS, RT-PCR, Western blotting analysis, invasion assay, transfection, to explore the molecular basis of NC-triggered anti-cancer activity. We found that NC inhibited cell growth, induced cell apoptosis, caused cell cycle arrest in ovarian cancer cells. Emerging evidence has demonstrated that Skp2 plays an important oncogenic role in ovarian cancer. Therefore, we also explored whether NC exerts its biologic function via downregulation of Skp2 in ovarian cancer cells. We observed that NC significantly inhibited the expression of Skp2 in ovarian cancer cells. Notably, overexpression of Skp2 abrogated the anti-cancer activity induced by NC in ovarian cancer cells. Consistently, downregulation of Skp2 expression enhanced the sensitivity of ovarian cancer cells to NC treatment. Thus, inactivation of Skp2 by NC could be a novel strategy for the treatment of human ovarian cancer.     

Establishment and characterization of two new human ovarian cancer cell lines UWOV1 and UWOV2 and a subline UWOV2 (Sf) growing in serum-free conditions: Growth characteristics,biochemical, and cytogenetic studies

Summary The establishment, growth, and characterization of two new continuously growing human ovarian cancer cell lines (UWOV1 and UWOV2) as, well as a subline (UWOV2, Sf) grown in chemically defined, serum-free medium are described. The cell lines were derived from ascitic tumors of two patients suffering from cystadenocarcinomas of the ovary. Both UWOV1 and UWOV2 lines grow in anchorage-dependent fashion as monolayers, whereas UWOV2 (Sf) forms multilayered domelike structures. Cytogenetic studies revealed nonrandom abnormalities involving chromosomes 1 and 11 in all three cell lines. Secretion of soluble collagen was detected in all three cell lines. In addition, UWOV2 (Sf) produces and secretes large amounts of extracellular matrix material with an ordered fibrillar structure which may function as an attachment factor for the serum-free cells. These cell lines seem to be useful for further studies of the biology of human ovarian cancer. This research was supported by grants from National Cancer Association (S. A.) and Bekker Trust Foundation.     

Reversing chemoresistance in cisplatin-resistant human ovarian cancer cells: a role of c-Jun NH2-terminal kinase 1

To investigate the role of activation of c-Jun NH2-terminal kinase 1 (JNK1) in mediating cisplatin-induced apoptosis and the possibility of induction of JNK activity in triggering relation to DNA damage and drug resistance. We investigated the difference of cisplatin-induced activation of JNK pathway and H2O2 alteration between cisplatin-sensitive human ovarian carcinoma cell line A2780 and its resistant variant A2780/DDP. JNK, p-JNK protein, and extracellular H2O2 levels were determined in both A2780 and A2780/DDP cells which were transfected with dominant negative allele of JNK and recombinant JNK1 separately. Both A2780 and A2780/DDP were treated with CDDP, the JNK pathway was activated and a prolonged JNK activation was maintained for at least 12 h in A2780, and only a transient activation (3 h) was detected in A2780/DDP in response to cisplatin treatment. Inhibition of JNK activity by transfection with a dominant negative allele of JNK blocked CDDP-induced apoptosis significantly in A2780 cells. Selective stimulation of the JNK pathway by lipofectamine-mediated delivery of recombinant JNK1 led to activation of c-Jun and decrease of extracellular H2O2, as well as apoptosis sensitization to CDDP in A2780/DDP cells. We concluded that JNK pathway might play an important role in mediating cisplatin-induced apoptosis in A2780 cells, and the duration of JNK activation might be critical in determining whether cells survive or undergo apoptosis. The resistance to CDDP can be reversed through activating c-Jun and decreasing extracellular generation of H2O2 by pcDNA3(FLAG)-JNK1-wt transfection in A2780/DDP cells.     

Prostasin may contribute to chemoresistance,repress cancer cells in ovarian cancer,and is involved in the signaling pathways of CASP/PAK2-p34/actin

Ovarian cancer is the deadliest of gynecologic cancers, largely due to the development of drug resistance in chemotherapy. Prostasin may have an essential role in the oncogenesis. In this study, we show that prostasin is decreased in an ovarian cancer drug-resistant cell line and in ovarian cancer patients with high levels of excision repair cross-complementing 1, a marker for chemoresistance. Our cell cultural model investigation demonstrates prostasin has important roles in the development of drug resistance and cancer cell survival. Forced overexpression of prostasin in ovarian cancer cells greatly induces cell death (resulting in 99% cell death in a drug-resistant cell line and 100% cell death in other tested cell lines). In addition, the surviving cells grow at a much lower rate compared with non-overexpressed cells. In vivo studies indicate that forced overexpression of prostasin in drug-resistant cells greatly inhibits the growth of tumors and may partially reverse drug resistance. Our investigation of the molecular mechanisms suggests that prostasin may repress cancer cells and/or contribute to chemoresistance by modulating the CASP/P21-activated protein kinase (PAK2)-p34 pathway, and thereafter PAK2-p34/JNK/c-jun and PAK2-p34/mlck/actin signaling pathways. Thus, we introduce prostain as a potential target for treating/repressing some ovarian tumors and have begun to identify their relevant molecular targets in specific signaling pathways.     

Clinical and prognostic effects of CDKN2A,CDKN2B and CDH13 promoter methylation in ovarian cancer: a study using meta-analysis and TCGA data

Abstract

Background: Promoter methylation of tumour suppressor genes (TSGs) CDKN2A, CDKN2B and CDH13 has been reported in ovarian cancer. However, the clinicopathological characteristics and prognostic role of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian carcinoma remained unclear.

Methods: The pooled odds ratio (OR) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated in this meta-analysis. The Cancer Genome Atlas data were obtained to confirm the role of CDKN2A, CDKN2B and CDH13 methylation in ovarian cancer.

Results: CDKN2A, CDKN2B and CDH13 promoter methylation was higher in ovarian cancer than in normal ovarian tissues. CDH13 promoter methylation was correlated with tumour histology (serous vs. non-serous type: OR?=?0.33, p?=?0.031). CDKN2A promoter methylation was not linked to overall survival (OS), but it was correlated with a poor prognosis in progression-free survival (HR?=?1.55, p?=?0.004). TCGA data showed no correlation between CDKN2A, CDKN2B and CDH13 methylation and OS as well as disease-free survival (DFS).

Conclusions: CDKN2A, CDKN2B and CDH13 promoter methylation may correlate with the increased risk of ovarian cancer. CDKN2A promoter methylation may be an independent prognostic biomarker for predicting progression-free survival.     

A new role for the P2X7 receptor: a scavenger receptor for bacteria and apoptotic cells in the absence of serum and extracellular ATP

The P2X7 receptor is widely recognized to mediate the proinflammatory effects of extracellular ATP. However this receptor in the absence of ATP may have a function unrelated to inflammation. Our data show that P2X7 expressed on the surface of monocyte/macrophages or on epithelial HEK-293 cells greatly augments the engulfment of latex beads and live and heat-killed bacteria by effector phagocyte in the absence of ATP and serum. The expression of P2X7 on the effector also confers the ability to phagocytose apoptotic target cells and an accumulation of P2X7 can be seen at the attachment point to the target. Activation of the P2X7 receptor by ATP causes a slow dissociation (over 10–15 min) of nonmuscle myosin from the P2X7 membrane complex and abolishes further P2X7-mediated phagocytosis of these targets. The recent crystal structure of the homologous zebrafish P2X4 receptor shows an exposed “nose” of the ectodomain (residues 115–162) which contains three of the five disulfide bonds conserved in all P2X receptors. Three short biotin-labeled peptides mimicking sequence of this exposed region bound to apoptotic target cells but not to either viable cells or to other target particles. All three peptides contained one or two cysteine residues and their replacement by alanine abolished peptide binding. These data implicate thiol-disulfide exchange reactions in the initial tethering of apoptotic cells to macrophage and establish P2X7 as one of the scavenger receptors involved in the recognition and removal of apoptotic cells in the absence of extracellular ATP and serum.     

Clinical significance of the NKG2D ligands,MICA/B and ULBP2 in ovarian cancer: high expression of ULBP2 is an indicator of poor prognosis

Objective  To investigate the clinical significance of the expression of the NKG2D ligands MICA/B and ULBP2 in ovarian cancer. Methods  Eighty-two ovarian cancer patients and six patients without ovarian cancer from Department of Obstetrics and Gynecology of Kyoto University Hospital were enrolled in this study between 1993 and 2003. Expression of MICA/B, ULBP2, and CD57 in ovarian cancer tissue and normal ovary tissue was evaluated by immunohistochemical staining, and the relationship of these results to relevant clinical patient data was analyzed. Expression of MICs, ULBP2, and HLA-class I molecules in 33 ovarian cancer cell lines and two normal ovarian epithelial cell lines, as well as levels of soluble MICs and ULBP2 in the culture supernatants, were measured. Results  Expression of MICA/B and ULBP2 was detected in 97.6 and 82.9% of ovarian cancer cells, respectively, whereas neither was expressed on normal ovarian epithelium. The expression of MICA/B in ovarian cancer was highly correlated with that of ULBP2. Strong expression of ULBP2 in ovarian cancer cells was correlated with less intraepithelial infiltration of T cells and bad prognoses for patients, suggesting that ULBP2 expression is a prognostic indicator in ovarian cancer. The expression of NKG2D ligands did not correlate with the levels of the soluble forms of the ligands. Conclusions  High expression of ULBP2 is an indicator of poor prognosis in ovarian cancer and may relate to T cell dysfunction in the tumor microenvironment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by grants from Grant-in-Aid for Scientific Research (19390426, 19591932, 18209052 and 19659421) from the Ministry of Education, Science, Sports, Culture and Technology of Japan.     

Familial breast/ovarian cancer and BRCA1/2 genetic screening: the role of immunohistochemistry as an additional method in the selection of patients.

Only 20-25% of families screened for BRCA1/2 mutations are found positive. Because only a positive result is informative, we studied the role of BRCA1/2 immunohistochemistry as an additional method for patient selection. From 53 high-risk-affected probands, 18 (34%) had available paraffin blocks of their tumors and were selected for this study. Mutation screening was done by conformation-sensitive gel electrophoresis and multiplex ligation-dependent probe amplification. For immunohistochemistry, 21 neoplastic specimens (15 breast carcinomas, 5 ovary neoplasms, and 1 rectal adenocarcinoma) were analyzed with BRCA1 (monoclonal antibody, Ab-1, oncogene) and BRCA2 (polyclonal antibody, Ab-2, oncogene) antibodies. Absence of the BRCA1 protein was confirmed in negative tumors by Western blotting. Seven patients were positive for BRCA1/2 mutations: 5 for BRCA1 and 2 for BRCA2. Four out of five positive patients had tumors negative for BRCA1 immunostaining, and the remaining 13 BRCA1-negative patients had positive BRCA1 immunostaining in all tumor samples. Sensitivity to predict for BRCA1 mutation carriers was 80%, and specificity was 100%, with a positive predictive value of 100% and a negative predictive value of 93%. This correlation was statistically significant (p=0.001). No correlation was observed for BRCA2. If larger studies confirm these results, high-risk patients with BRCA1-negative tumors should be screened first for this gene.     

The significance of an increase in soluble interleukin-2 receptor level in colorectal cancer and its biological regulating role in the physiological switching of the immune response cytokine network from TH1 to TH2 and back

 Current research has still not clarified the biological role of soluble interleukin(IL)-2 receptor (sIL-2R) and the significance of its increase in the serum of colon cancer patients compared to healthy subjects. To address these questions at the immunological level in a group of patients and healthy subjects, we determined the sIL-2R level in the serum and its release from peripheral blood mononuclear cells (PBMC) as a function of tumour necrosis factor (TNF) α, IL-1α, IL-1β, IL-2, interferon (IFN) γ, IL-4, IL-6 and IL-10 levels in the serum and PBMC production; and PBMC proliferative responses to IL-2, IL-4 and anti-CD3 monoclonal antibody (CD3), variously combined. The level of sIL-2R in patients’ serum was higher than in healthy subjects and correlated with the stage of advancement. Moreover, while in healthy subjects the serum level of sIL-2R was not significantly correlated with other parameters, in patients it was positively related to IL-4, IL-6 and IL-10 serum levels, PBMC IL-4 production and to the PBMC proliferative response to CD3 and CD3+IL-2; it was negatively correlated to IL-2 serum level and IL-1β PBMC release. A negative connection between IFNγ serum level and the PBMC production of sIL-2R was also found. This suggests that the increase of sIL-2R in the serum of patients, compared to healthy subjects, is involved in the inappropriate expansion of the T helper (TH2) suppressive immune response, which we previously reported. The multivariate statistical method supported the above suggestions and we also found that, in healthy subjects, the up- and down-regulation of sIL-2R in the serum within the physiological ranges seems to have a regulating role in the relationships between TNFα, IFNγ and IL-4, IL-6, contributing to the operation of the cytokine network between TH1 and TH2 cells. However, in patients compared to healthy subjects the increased sIL-2R serum level seems to direct the immune response towards a suppressive type, which may be due to an alteration in the above-mentioned physiological regulating role. Received: 12 April 1997 / Accepted: 4 September 1997     

Evaluation of the potential therapeutic role of a new generation of vitamin D analog,MART-10, in human pancreatic cancer cells in vitro and in vivo

Pancreatic cancer is a lethal disease with no known effective chemotherapy and radiotherapy, and most patients are diagnosed in the late stage, making them unsuitable for surgery. Therefore, new therapeutic strategies are urgently needed. 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] is known to possess antitumor actions in many cancer cells in vitro and in vivo models. However, its clinical use is hampered by hypercalcemia. In this study, we investigated the effectiveness and safety of a new generation, less calcemic analog of 1α,25(OH)₂D₃, 19-nor-2α-(3-hydroxypropyl)-1α,25-dihydroxyvitamin D₃ (MART-10), in BxPC-3 human pancreatic carcinoma cells in vitro and in vivo. We demonstrate that MART-10 is at least 100-fold more potent than 1α,25(OH)₂D₃ in inhibiting BxPC-3 cell proliferation in a time- and dose-dependent manner, accompanied by a greater upregulation of cyclin-dependent kinase inhibitors p21 and p27 and a greater downregulation of cyclin D3 and cyclin-dependent kinases 4 and 5, leading to a greater increase in the fraction of cells in G₀/G₁ phase. No induction of apoptosis and no effect on Cdc25 phosphatases A and C were observed in the presence of either MART-10 or 1α,25(OH)₂D₃. In a xenograft mouse model, treatment with 0.3 µg/kg body weight of MART-10 twice/week for 3 weeks caused a greater suppression of BxPC-3 tumor growth than the same dose of 1α,25(OH)₂D₃ without inducing hypercalcemia and weight loss. In conclusion, MART-10 is a promising agent against pancreatic cancer growth. Further clinical trial is warranted.     

N-myristoyltransferase 2 expression in human colon cancer: cross-talk between the calpain and caspase system

A number of viral and eukaryotic proteins which undergo a lipophilic modification by the enzyme N-myristoyltransferase (NMT: NMT1 and NMT2) are required for signal transduction and regulatory functions. To investigate whether NMT2 contributes to the pathogenesis of colorectal carcinoma, we observed a higher expression of NMT2 in most of the cases of cancerous tissues compared to normal tissues (84.6% of cases; P < 0.05) by Western blot analysis. Furthermore, protein-protein interaction of NMTs revealed that m-calpain interacts with NMT1 while caspase-3 interacts with NMT2. Our findings provide the first evidence of higher expression of NMT2 in human colorectal adenocarcinomas and the interaction of both forms of NMT with various signaling molecules.     

Potential roles and targeted therapy of the CXCLs/CXCR2 axis in cancer and inflammatory diseases

The chemokine receptor CXCR2 and its ligands are implicated in the progression of tumours and various inflammatory diseases. Activation of the CXCLs/CXCR2 axis activates multiple signalling pathways, including the PI3K, p38/ERK, and JAK pathways, and regulates cell survival and migration. The CXCLs/CXCR2 axis plays a vital role in the tumour microenvironment and in recruiting neutrophils to inflammatory sites. Extensive infiltration of neutrophils during chronic inflammation is one of the most important pathogenic factors in various inflammatory diseases. Chronic inflammation is considered to be closely correlated with initiation of cancer. In addition, immunosuppressive effects of myeloid-derived suppressor cells (MDSCs) against T cells attenuate the anti-tumour effects of T cells and promote tumour invasion and metastasis. Over the last several decades, many therapeutic strategies targeting CXCR2 have shown promising results and entered clinical trials. In this review, we focus on the features and functions of the CXCLs/CXCR2 axis and highlight its role in cancer and inflammatory diseases. We also discuss its potential use in targeted therapies.