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Objectives Lymphocyte-activation gene 3 (LAG-3) represents a potential immune checkpoint target for cancer treatment. We investigated LAG-3 expression and its prognostic value in patients with surgically treated clear cell renal cell carcinoma (RCC) and correlated LAG-3 expression with programmed cell death ligand 1(PD-L1). Methods We evaluated LAG-3 and PD-L1 expression using immunohistochemistry on tissue microarrays incorporating 134 primary excision specimens of clear cell RCC (ccRCC). The patients were analyzed as two groups: the whole cohort and those with metastatic RCC (mRCC). The cancer genome atlas (TCGA) data analysis of LAG-3 was done through UALCAN web servers. Results Using the UALCAN cancer transcriptional data analysis, we found that LAG-3 was overexpressed in ccRCC. LAG-3 expression was significantly correlated with PD-L1 expression in the whole cohort and in the mRCC group (all, p < 0.05). Both LAG-3⁺ RCC and PD-L1⁺ RCC presented with a higher TNM stage and higher Fuhrman nuclear grade (all, p < 0.05). PD-L1⁺/LAG-3⁺ RCC and PD-L1⁻/LAG-3⁺ RCC showed poorer cancer-specific survival (CSS) than PD-L1⁻/LAG-3⁻ RCC (all, p = 0.01). Similarly, PD-L1⁺/LAG-3⁺ mRCC and PD-L1⁻/LAG-3⁺ mRCC showed poorer CSS than PD-L1⁻/LAG-3⁻ mRCC (all, p < 0.05). Multivariate analysis showed that PD-L1⁺/LAG-3⁺ mRCC (hazard ratio: 3.19; 95% CI: 0.77–13.67; p = 0.033) was a predictor of poor CSS. Conclusion Both LAG-3⁺ and PD-L1⁺ RCC have adverse pathological features, and their coexpression predicts worse clinical outcomes. Our findings suggest LAG-3 blockade in combination with programmed cell death 1/PD-L1 blockade as a potential therapeutic approach for RCC.

Lymphocyte-activation gene 3 (LAG-3) protein is a potential immune checkpoint target for cancer treatment. Thus, we investigated LAG-3 expression and its prognostic value in clear cell renal cell carcinoma (ccRCC) and correlated LAG-3 expression with programmed cell death ligand 1 (PD-L1) using immunohistochemistry on tissue microarrays incorporating 134 ccRCC primary excision specimens. The study patients were analyzed as two groups: the whole cohort and those with metastatic renal cell carcinoma (mRCC). LAG-3 expression was significantly correlated with PD-L1 expression in the whole cohort and in the mRCC group (all, p p < 0.05). According to the International Metastatic Renal Cell Carcinoma Database Consortium risk classification, there were more intermediate- and poor-risk patients in both LAG-3⁺ mRCC and PD-L1⁺ mRCC than LAG-3⁻ mRCC and PD-L1⁻ mRCC (all, p < 0.05). PD-L1⁺/LAG-3⁺ RCC and PD-L1⁻/LAG-3⁺ RCC in the whole cohort showed poorer cancer-specific survival (CSS) than PD-L1⁻/LAG-3⁻ RCC (all, p = 0.01). Similarly, PD-L1⁺/LAG-3⁺ mRCC and PD-L1⁻/LAG-3⁺ mRCC showed poorer CSS than PD-L1⁻/LAG-3⁻ mRCC (all, p < 0.05). Multivariate analysis showed that PD-L1⁺/LAG-3⁺ mRCC (hazard ratio: 3.19; p = 0.033) was a predictor of poor CSS. Our findings provided a scientific rationale for LAG-3 blockade in combination with programmed cell death 1/PD-L1 blockade as a potential therapeutic approach for RCC.

Hypoxic placentation has been considered as a key step for the development of preeclampsia (PE); however, the underlying epigenetic mechanisms are still not fully understood. The purpose of this study is to investigate the whole genome DNA methylation status of PE. A microarray analysis using the Infinium HumanMethylation450 BeadChip assay in the placentas and maternal peripheral blood (PB) from PE patients and normal controls was performed. For validation, a quantitative RT-PCR analysis was used. Maternal PB showed 71 differentially methylated CpG loci (44 hypermethylated and 27 hypomethylated), while placenta revealed 365 loci (37 hypermethylated and 328 hypomethylated) at the statistical significance level of |Δβ| ≥ 0.17 and P ≤ 0.01. Notably, among the candidates showing significant signals, GRK5 (a member of G protein-coupled receptor kinase family that has previously been known to be associated with PE) showed a significantly hypomethylated level in the placentas of PE patients (Δβ = −0.176, P = 2.8 × 10−5). In the validation for the potential effect of GRK5 methylation on the gene regulation, GRK5 expression was significantly increased in the placentas from PE patients compared to those from controls (P = 0.027). In further GO analysis, genes of MHC class II protein complex showed the most significant differential methylation in the maternal PB of PE patients, while genes of palate development were differentially methylated in the placenta. Although further replication and functional studies are required, our preliminary results suggest that PE has distinct DNA methylation profiles in the maternal PB and placentas, which may provide insight into future research.

Background: Arsenic compounds (As2O3 and As4O6) have demonstrated anticancer effects in various malignancies. In this study, the cytotoxicity of arsenic compounds on ovarian cancer cell lines and the anticancer activity of the combination of arsenic compounds and cisplatin IN chemoresistant ovarian cancer cells were investigated. Methods: We investigated the cytotoxicity of As2O3 and As4O6 and their combinations with cisplatin in the paclitaxel-sensitive ovarian cancer cell lines SKOV3ip1 and HeyA8 and paclitaxel-resistant ovarian cancer cell lines SKOV3TRip2 and HeyA8-MDR. Growth and apoptosis were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. For detection of apoptotic cells, immunofluorescence was performed using a cleaved caspase-3 antibody. Cell-cycle distribution was determined by propidium iodide staining and flow cytometry. Results: Treatment of each cell line with As2O3 or As4O6 led to a marked dose-dependent inhibition of cell growth. As2O3 and As4O6 treatment induced caspase-3-dependent apoptosis in all cell lines compared to the respective control groups (p 2O3 and As4O6 induced apoptosis of paclitaxel-sensitive and -resistant cancer cell lines following G2/M cell cycle arrest (p 2O3 or As4O6 in the paclitaxel-resistant ovarian cancer cell lines. Conclusions: As2O3 and As4O6 can inhibit cell growth and induce apoptosis in paclitaxel-sensitive and -resistant ovarian cancer cell lines. Their combination with cisplatin resulted in a synergistic effect in paclitaxel-resistant cancer cell lines. These results suggest that arsenic compounds may be given in monotherapy or combination therapy with cisplatin for treating paclitaxel-resistant ovarian cancer.

Endometrial cancer is the sixth most common cancer in women worldwide. Peroxiredoxins (PRDXs) are antioxidant enzymes that serve important roles in cell differentiation, proliferation, and apoptosis. In the present study, the potential associations between PRDX expression and endometrial cancer were investigated. The expression levels of various PRDX mRNAs were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) in endometrial cancer tissues (n=26) and normal endometrial tissues (n=10). Additionally, the expression of PRDX isoforms was immunohistochemically examined in endometrial cancer tissues and adjacent normal endometrial tissues from 42 patients. Finally, the associations between high PRDX expression levels and clinicopathological features were examined in patients with endometrial cancer. Analysis of PRDX expression in endometrial cancer tissues and normal endometrial tissues by semi-quantitative RT-PCR showed that all PRDX isoforms had increased expression in the endometrial cancer tissues compared with that in the normal endometrium, and the differences in the expression levels of PRDX1 and PRDX3 between cancer and normal tissues were statistically significant (P=0.0015 and P=0.0134, respectively). Additionally, analysis of PRDX expression in endometrial cancer and paired normal endometrial tissues by immunohistochemistry showed strong cytoplasmic staining of PRDX3 and PRDX5 in cancer tissues, with high PRDX3 (25/42, 59.5%) and PRDX5 (32/42, 76.2%) appearing more frequently in endometrial cancer than in normal endometrial tissues (P=0.0001 and P=0.0023, respectively). Furthermore, high expression of PRDX5 was associated with advanced-stage endometrial cancer (P=0.0399). Although the 5-year survival rate was marginally higher in patients with low expression of PRDX3 and PRDX5, this result was not statistically significant. In summary, PRDX3 and PRDX5 are highly expressed in endometrial cancer and could be associated with advanced stage and poor prognosis. Therefore, these proteins may potentially be used as prognostic markers for endometrial cancer.

Tetraarsenic oxide (As4O6, TAO) is a new arsenic compound that inhibits cell growth and induces apoptosis in human cervical cancer cell lines. In the present study, we report that the growth of tumor cells (CaSki) was inhibited by treatment with TAO alone or in combination with cisplatin or paclitaxel in vitro and in vivo. Proliferation was assessed by WST-1 assay, and apoptosis was assessed by Annexin-V/PI FACS analysis in the CaSki cell line treated with a single agent or with the combinations of two agents. Expression of apoptosis-related proteins was analyzed by western blot analysis. A mouse xenograft model using CaSki cells was used to determine the in vivo activity of tetraarsenic oxide alone and in combination with cisplatin or paclitaxel by estimation of tumor size. At the end of the experiment, tumor tissue from each mouse was removed and processed for TUNEL analysis for confirmation of apoptotic cells. TAO was able to inhibit cell proliferation in a time- and dose-dependent manner. A combination of TAO and cisplatin effectively induced apoptosis by activating caspase-3. Using a mouse xenograft model, the sizes of tumors which were treated with a single agent and with a combination of agents decreased in a time-dependent manner. A combination of TAO and cisplatin resulted in a significantly reduced tumor size (P

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.

Objective: To compare plasma protein expression between patients with squamous cell carcinoma (SCC) of the cervix and normal controls. Methods: Plasma samples from patients with benign gynecological disease (normal cervix, n=6) and cervical cancer (SCC, n=6) were subjected to plasma proteomic analysis using two dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS). Western blotting and immunoturbidimetric assay were performed to validate the results of 2-DE. Results: Eight proteins showed differential expression between controls and SCC patients; six (ceruloplasmin, complement C3, afamin precursor, alpha-1-B-glycoprotein, transferrin, alpha-fibrinogen precursor) were up-regulated, while two (chain A, crystal structure of antithrombin and apolipoprotein A-IV precursor) were down-regulated in the plasma of SCC patients. Western blotting analysis revealed significant elevation of ceruloplasmin, complement C3, afamin, and alpha-1-B-glycoprotein in the plasma of SCC patients in comparison to controls. Immunoturbidimetric assay of a larger group confirmed the results of 2-DE and Western blotting, and showed that ceruloplasmin and complement C3 were significantly elevated in the plasma of SCC patients in comparison with controls and patients with carcinoma in situ (CIS) of the uterine cervix. Conclusion: Plasma protein expression determined using 2-DE and MALDI-MS will give a chance to identify tumor-specific biomarkers for SCC of the cervix.