A novel biological antibacterial polyvinyl alcohol/polyionic liquid hydrogel for wound dressing.

The release of antibiotics or anions by traditional bacteriostatic agents led to the development of bacterial drug resistance and environmental pollution. Ionic liquids (ILs) have become important choices for antibacterial agents because of their excellent physical, chemical and biological properties. In this paper, the bioactivities of 1-vinyl-3-butylimidazolium chloride ([VBIM]Cl, IL) and poly (1-vinyl-3-butylimidazolium chloride) (P[VBIM]Cl, PIL) were evaluated, and the potential antibacterial material was used to synthesize hydrogels. Using the colony formation assay and the Oxford cup method, antibacterial effect of IL and PIL were tested. Cell-Counting-Kit-8 (CCK-8) experiments were used to study the IC50 (half maximal inhibitory concentration) values of IL and showed 1.47 mg/mL, 0.35 mg/mL and 0.33 mg/mL at 24 h, 48 h and 72 h, respectively. The IC50 value of PIL were 12.15 µg/mL, 12.06 µg/mL and 11.76 µg/mL at 24 h, 48 h and 72 h, respectively. The PIL is further crosslinked with polyvinyl alcohol (PVA) to form a novel hydrogel through freeze-thaw cycles. The newly fabricated hydrogel exhibited a high water content, excellent water absorption properties and outstanding mechanical performance. Using the colony formation assay and the inhibition zone assay, the hydrogels exhibited favorable antibacterial effects (against E.coli and S.aureus) such that nearly 100% of the bacteria were killed in liquid medium while cultivating with H4 (synthesized by 0.5 g PIL and 1g PVA). In addition, the cytotoxicity of PIL was significantly reduced through hydrogen bond crosslinking. H4 showed the highest antibacterial activity and a good biocompatibility. The results indicated that the PVA&PIL hydrogels had great potential for wound dressing.

Methylation of protocadherin 10, a novel tumor suppressor, is associated with poor prognosis in patients with gastric cancer.

BACKGROUND & AIMS: By using methylation-sensitive representational difference analysis, we identified protocadherin 10 (PCDH10), a gene that encodes a protocadherin and is silenced in a tumor-specific manner. We analyzed its epigenetic inactivation, biological effects, and prognostic significance in gastric cancer. METHODS: Methylation status was evaluated by combined bisulfite restriction analysis and bisulfite sequencing. The effects of PCDH10 re-expression were determined in growth, apoptosis, proliferation, and invasion assays. PCDH10 target genes were identified by complementary DNA microarray analysis. RESULTS: PCDH10 was silenced or down-regulated in 94% (16 of 17) of gastric cancer cell lines; expression levels were restored by exposure to demethylating agents. Re-expression of PCDH10 in MKN45 gastric cancer cells reduced colony formation in vitro and tumor growth in mice; it also inhibited cell proliferation (P < .01), induced cell apoptosis (P < .001), and repressed cell invasion (P < .05), up-regulating the pro-apoptosis genes Fas, Caspase 8, Jun, and CDKN1A; the antiproliferation gene FGFR; and the anti-invasion gene HTATIP2. PCDH10 methylation was detected in 82% (85 of 104) of gastric tumors compared with 37% (38 of 104) of paired nontumor tissues (P < .0001). In the latter, PCDH10 methylation was higher in precancerous lesions (27 of 45; 60%) than in chronic gastritis samples (11 of 59; 19%) (P < .0001). After a median follow-up period of 16.8 months, multivariate analysis revealed that patients with PCDH10 methylation in adjacent nontumor areas had a significant decrease in overall survival. Kaplan-Meier survival curves showed that PCDH10 methylation was associated significantly with shortened survival in stage I-III gastric cancer patients. CONCLUSIONS: PCDH10 is a gastric tumor suppressor; its methylation at early stages of gastric carcinogenesis is an independent prognostic factor.

Epigenetic identification of ubiquitin carboxyl-terminal hydrolase L1 as a functional tumor suppressor and biomarker for hepatocellular carcinoma and other digestive tumors.

UNLABELLED: The ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) is a carboxyl-terminal ubiquitin hydrolase regulating cellular ubiquitin levels, recently suggested as a tumor suppressor. However, the role of UCHL1 in hepatocellular carcinoma (HCC) is not clear. We investigated the expression and DNA methylation of the UCHL1 in primary HCC, liver metastases from digestive carcinomas, and primary digestive cancers. UCHL1 is expressed in all normal tissues and immortalized normal epithelial cell lines, but was low or silenced in 77% (10/13) of HCC cell lines, which is well correlated with its promoter methylation status. Methylation was further detected in 44% (12/27) of HCCs, but less in metastatic tumors generated from colorectal and stomach in the liver (19%, 3/16; P < 0.05). Methylation was also detected in primary digestive tumors, including 71% (22/31) of colon, 77% (53/69) of gastric, and 40% (18/45) of esophageal carcinomas, but none or occasionally in paired adjacent nontumor tissues. Detailed methylation analysis of 49 CpG sites at a 540-bp promoter region by bisulfite genomic sequencing confirmed the methylation. UCHL1 silencing could be reversed by chemical or genetic demethylation of the promoter, indicating direct epigenetic silencing. Restoring UCHL1 expression in silenced cell lines significantly inhibited their growth and colony formation ability by inhibiting cell proliferation, causing cell cycle arrest in G2/M phase and inducing apoptosis through the intrinsic caspase-dependent pathway. Moreover, UCHL1 directly interacts with p53 and stabilizes p53 through the ubiquitination pathway. CONCLUSION: Epigenetic inactivation of UCHL1 is common in primary HCCs and other digestive tumors. UCHL1 appears to be a functional tumor suppressor involved in the tumorigenesis of HCCs and other digestive cancers.

Genomic stratification and liquid biopsy in a rare adrenocortical carcinoma (ACC) case, with dual lung metastases.

Adrenocortical carcinoma is a rare malignancy with a poor prognosis and few treatment options. Molecular characterization of this cancer remains limited. We present a case of an adrenocortical carcinoma (ACC) in a 37-yr-old female, with dual lung metastases identified 1 yr following commencement of adjuvant mitotane therapy. As standard therapeutic regimens are often unsuccessful in ACC, we undertook a comprehensive genomic study into this case to identify treatment options and monitor disease progress. We performed targeted and whole-genome sequencing of germline, primary tumor, and both metastatic tumors from this patient and monitored recurrence over 2 years using liquid biopsy for ctDNA and steroid hormone measurements. Sequencing revealed the primary and metastatic tumors were hyperhaploid, with extensive loss of heterozygosity but few structural rearrangements. Loss-of-function mutations were identified in MSH2, TP53, RB1, and PTEN, resulting in tumors with mismatch repair signatures and microsatellite instability. At the cellular level, tumors were populated by mitochondria-rich oncocytes. Longitudinal ctDNA mutation and hormone profiles were unable to detect micrometastatic disease, consistent with clinical indicators of disease remission. The molecular signatures in our ACC case suggested immunotherapy in the event of disease progression; however, the patient remains free of cancer. The extensive molecular analysis presented here could be applied to other rare and/or poorly stratified cancers to identify novel or repurpose existing therapeutic options, thereby broadly improving diagnoses, treatments, and prognoses.

A link between the fibroblast growth factor axis and the miR-16 family reveals potential new treatment combinations in mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with very limited therapeutic options. Fibroblast growth factor (FGF) signals play important roles in mesothelioma cell growth. Several FGFs and FGF receptors (FGFRs) are predicted targets of the miR-15/16 family, which is downregulated in MPM. The aim of this study was to explore the link between the miR-15/16 family and the FGF axis in MPM. Expression analyses via RT-qPCR showed downregulation of the FGF axis after transfection with miR-15/16 mimics. Direct interaction was confirmed by luciferase reporter assays. Restoration of miR-15/16 led to dose-dependent growth inhibition in MPM cell lines, which significantly correlated with their sensitivity to FGFR inhibition. Treatment with recombinant FGF2 prevented growth inhibition and further reduced the levels of FGF/R-targeting microRNAs, indicating a vicious cycle between miR-15/16 down- and FGF/FGFR signaling upregulation. Combined inhibition of two independent miR-15/16 targets, the FGF axis and Bcl-2, resulted in additive or synergistic activity. Our data indicate that post-transcriptional repression of FGF-mediated signals contributes to the tumor suppressor function of the microRNA-15/16 family. Inhibiting hyperactivated FGF signals and Bcl-2 might serve as a novel therapeutic combination strategy in MPM.

Dysregulated Expression of the MicroRNA miR-137 and Its Target YBX1 Contribute to the Invasive Characteristics of Malignant Pleural Mesothelioma.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an aggressive malignancy linked to asbestos exposure. On a genomic level, MPM is characterized by frequent chromosomal deletions of tumor suppressors, including microRNAs. MiR-137 plays a tumor suppressor role in other cancers, so the aim of this study was to characterize it and its target Y-box binding protein 1 (YBX1) in MPM. METHODS: Expression, methylation, and copy number status of miR-137 and its host gene MIR137HG were assessed by polymerase chain reaction. Luciferase reporter assays confirmed a direct interaction between miR-137 and Y-box binding protein 1 gene (YBX1). Cells were transfected with a miR-137 inhibitor, miR-137 mimic, and/or YBX1 small interfering RNA, and growth, colony formation, migration and invasion assays were conducted. RESULTS: MiR-137 expression varied among MPM cell lines and tissue specimens, which was associated with copy number variation and promoter hypermethylation. High miR-137 expression was linked to poor patient survival. The miR-137 inhibitor did not affect target levels or growth, but interestingly, it increased miR-137 levels by means of mimic transfection suppressed growth, migration, and invasion, which was linked to direct YBX1 downregulation. YBX1 was overexpressed in MPM cell lines and inversely correlated with miR-137. RNA interference-mediated YBX1 knockdown significantly reduced cell growth, migration, and invasion. CONCLUSIONS: MiR-137 can exhibit a tumor-suppressive function in MPM by targeting YBX1. YBX1 knockdown significantly reduces tumor growth, migration, and invasion of MPM cells. Therefore, YBX1 represents a potential target for novel MPM treatment strategies.

Exploring Mechanisms of MicroRNA Downregulation in Cancer.

MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression at a posttranscriptional level. Each miRNA controls the expression of multiple messenger RNAs (mRNAs) and their dysregulation has been implicated in multiple cancer phenotypes. While some miRNAs are upregulated, global downregulation of miRNA expression is often the rule in cancer. A multitude of potential mechanisms drive aberrant miRNA expression in cancer; miRNA coding regions can harbour genomic defects including mutations, amplifications or deletions, and some miRNAs are broadly repressed by transcription factors such as Myc or have epigenetic modifications to their promoter regions such as hypermethylation of CpG islands. Additionally, the suppression of components of the miRNA processing machinery has been shown to reduce mature miRNA expression and contribute to the malignant phenotype. Understanding the mechanisms driving miRNA downregulation is important in uncovering the critical and complex role of miRNAs in cancer biology. This review will outline the multiple mechanisms by which cancer cells suppress miRNA expression.

Promoter methylation of the Wnt/beta-catenin signaling antagonist Dkk-3 is associated with poor survival in gastric cancer.

BACKGROUND: Abnormal activation of the Wnt/beta-catenin signaling pathway is common and critical in the pathogenesis of digestive cancers. In this study, the authors investigated the promoter methylation of the dickkopf homolog 3 gene Dkk-3 in these cancers and its prognostic significance in gastric cancer. METHODS: Dkk-3 methylation was assessed in 173 patients with gastric cancers (including 104 patients who were followed for up to 4090 days) and in 128 patients with colorectal cancer. Cell growth was evaluated by using a colony-formation assay. For survival analyses, the authors used Kaplan-Meier plots, the log-rank test, and Cox proportional regression. RESULTS: Dkk-3 was silenced or down-regulated in 12 of 17 gastric cancer cell lines (70.6%) and in 3 of 9 colon cancer cell lines (33.3%). The loss of gene expression was associated with promoter methylation, which could be restored by demethylating agents. Ectopic expression of Dkk-3 suppressed colony formation. Moreover, methylation of Dkk-3 was detected in 117 of 173 primary gastric tumors (67.6%) and in 67 of 128 colorectal tumors (52.3%). The clinical significance and the prognostic value of Dkk-3 methylation also were examined in 104 gastric cancers and in 84 colorectal cancers. Multivariate analysis indicated that Dkk-3 methylation was associated significantly and independently with poor disease survival (relative risk, 2.534; 95% confidence interval, 1.54-4.17; P=.002) in gastric cancer, but not in colorectal cancer. Kaplan-Meier survival curves revealed that patients who had Dkk-3 methylated gastric cancers had a significantly shorter survival (median, 0.76 years) compared with patients who did not have Dkk-3 methylation (median, 2.68 years; P<.0001; log-rank test). CONCLUSIONS: Epigenetic silencing of the Dkk-3 gene by promoter methylation was a common event in gastric cancer and was associated with a poor outcome in such patients.

Cytochemical and ultrastructural changes in the osteoclast-like giant cells of giant cell tumor of bone following bisphosphonate administration.

Giant cell tumor of bone (GCT) is a local aggressive neoplasm of bone characterized by expansive osteolytic lesions at the epiphysis of long bones. Bisphosphonates have been used to prevent bone resorption in secondary osteolytic tumors because of their strong anti-osteoclastic action. The authors studied the apoptosis and ultrastructural changes induced in osteoclast-like giant cells of GCT, following treatment with the aminobisphosphonate pamidronate in 16 patients with GCT of bone. Transmission electron microscopy (TEM) was used to identify ultrastructural changes, indicative of apoptosis, in the cytoplasm and the nucleus of the giant cells. Significant changes were observed in tumor samples from all 16 patients. In the cytoplasm these changes were characterized by abundant large tubular vesicles containing a central electrodense core scattered through the cytoplasm. In addition, mitochondria in the sections from pamidronate-treated patients appeared to be edematous when compared with sections from untreated patients. Nuclear changes in the giants cells were characterized by the formation of dense chromatin material scattered throughout the nucleus. The TUNEL labeling assay indicated that the mean pretreatment apoptotic index of 7.8% increased to 53% following pamidronate treatment. This was statistically significant (p<.001) and correlated well with the ultrastructural changes noted on TEM. The formation of abundant tubular vesicles in giant cells following bisphosphonate treatment may reflect disturbed vesicular trafficking and may affect the bone resorbing activity of giant cells.