Risk factors for diabetic foot ulcers in metreleptin naïve patients with lipodystrophy.

AIM: Patients with lipodystrophy are at high risk for chronic complications of diabetes. Recently, we have reported 18 diabetic foot ulcer episodes in 9 subjects with lipodystrophy. This current study aims to determine risk factors associated with foot ulcer development in this rare disease population. METHODS: Ninety metreleptin naïve patients with diabetes registered in our national lipodystrophy database were included in this observational retrospective cohort study (9 with and 81 without foot ulcers). RESULTS: Patients with lipodystrophy developing foot ulcers had longer diabetes duration (p = 0.007), longer time since lipodystrophy diagnosis (p = 0.008), and higher HbA1c levels (p = 0.041). Insulin use was more prevalent (p = 0.003). The time from diagnosis of diabetes to first foot ulcer was shorter for patients with generalized lipodystrophy compared to partial lipodystrophy (p = 0.036). Retinopathy (p < 0.001), neuropathy (p < 0.001), peripheral artery disease (p = 0.001), and kidney failure (p = 0.003) were more commonly detected in patients with foot ulcers. Patients with foot ulcers tended to have lower leptin levels (p = 0.052). Multiple logistic regression estimated significant associations between foot ulcers and generalized lipodystrophy (OR: 40.81, 95% CI: 3.31-503.93, p = 0.004), long-term diabetes (≥ 15 years; OR: 27.07, 95% CI: 2.97-246.39, p = 0.003), and decreased eGFR (OR: 13.35, 95% CI: 1.96-90.67, p = 0.008). CONCLUSIONS: Our study identified several clinical factors associated with foot ulceration among patients with lipodystrophy and diabetes. Preventive measures and effective treatment of metabolic consequences of lipodystrophy are essential to prevent the occurrence of foot ulcers in these high-risk individuals.

Extracellular vesicle-mediated suicide mRNA/protein delivery inhibits glioblastoma tumor growth in vivo.

Extracellular vesicles (EVs) are considered as important mediators of intercellular communication, which carry a diverse repertoire of genetic information between cells. This feature of EVs can be used and improved to advance their therapeutic potential. We have previously shown that genetically engineered EVs carrying the suicide gene mRNA and protein-cytosine deaminase (CD) fused to uracil phosphoribosyltransferase (UPRT)-inhibited schwannoma tumor growth in vivo. To further examine whether this approach can be applied to other cancer types, we established a subcutaneous xenograft glioblastoma tumor model in mice, as glioblastoma represents the most common primary brain tumor, which is highly aggressive compared with the original schwannoma tumor model. U87-MG glioblastoma cells were implanted into the flanks of nude SCID mice, and the animals were intratumorally injected with the EVs isolated from the cells expressing EGFP or CD-UPRT. After the intraperitoneal administration of the prodrug 5-fluorocytosine, the tumor growth was assessed by regular caliper measurements. Our data revealed that the treatment with the CD-UPRT-enriched EVs significantly reduced the tumor growth in mice. Taken together, our findings suggest that EVs uploaded with therapeutic CD-UPRT mRNA/protein may be a useful tool for glioblastoma treatment.

miR-200a-mediated suppression of non-muscle heavy chain IIb inhibits meningioma cell migration and tumor growth in vivo.

miR-200a has been implicated in the pathogenesis of meningiomas, one of the most common central nervous system tumors in humans. To identify how miR-200a contributes to meningioma pathogenesis at the molecular level, we used a comparative protein profiling approach using Gel-nanoLC-MS/MS and identified approximately 130 dysregulated proteins in miR-200a-overexpressing meningioma cells. Following the bioinformatic analysis to identify potential genes targeted by miR-200a, we focused on the non-muscle heavy chain IIb (NMHCIIb), and showed that miR-200a directly targeted NMHCIIb. Considering the key roles of NMHCIIb in cell division and cell migration, we aimed to identify whether miR-200a regulated these processes through NMHCIIb. We found that NMHCIIb overexpression partially rescued miR-200a-mediated inhibition of cell migration, as well as cell growth in vitro and in vivo. Moreover, siRNA-mediated silencing of NMHCIIb expression resulted in a similar migration phenotype in these cells and inhibited meningioma tumor growth in mice. Taken together, these results suggest that NMHCIIb might serve as a novel therapeutic target in meningiomas.

Depletion of minichromosome maintenance protein 7 inhibits glioblastoma multiforme tumor growth in vivo.

Minichromosome maintenance (MCM) proteins are key elements that function as a part of the pre-replication complex to initiate DNA replication in eukaryotes. Consistent with their roles in initiating DNA replication, overexpression of MCM family members has been observed in several malignancies. Through bioinformatic analysis of The Cancer Genome Atlas's data on glioblastoma multiforme (GBM), we found that the genomic region containing MCM7 gene was amplified in more than 80% of the present cases. To validate this finding and to identify the possible contribution of the remaining members of the MCM family to GBM progression, we used quantitative real-time PCR to analyze the gene expression profiles of all MCM family members in Grade IV (GBM) tissue samples and observed a significant upregulation in GBM samples compared with normal white matter tissues. In addition, we compared the observed gene expression profiles with those of Grade II and Grade III astrocytoma samples and determined that the observed upregulation was restricted and specific to Grade IV. MCM7 was the most upregulated gene in the gene set we analyzed, and therefore we wanted to identify the role of MCM7 in GBM progression. We determined that siRNA-mediated knockdown of MCM7 expression reduced GBM cell proliferation and also inhibited tumor growth in both xenograft and orthotopic mouse models of GBM. Taken together, our data suggest that MCM7 can be a potential prognostic marker and a novel therapeutic target in GBM therapy.

HSV-1 amplicon-mediated post-transcriptional inhibition of Rad51 sensitizes human glioma cells to ionizing radiation.

Standard treatment for glioblastoma multiforme and other brain tumors consists of surgical resection followed by combined radio-/chemotherapy. However, radiation resistance of tumor cells limits the success of this treatment, and the tumors invariably recur. Therefore, the selective inhibition of molecular mediators of radiation resistance may provide therapeutic benefit to the patient. One of these targets is the Rad51 protein, which is a key component of the homologous recombinational repair of DNA double-strand breaks. Here, we investigated whether post-transcriptional silencing of Rad51 by herpes simplex virus-type 1 (HSV-1) amplicon vector-mediated short interfering RNA expression can enhance the antitumor effect of radiation therapy. We demonstrate that these vectors specifically and efficiently inhibited the radiation-induced recruitment of Rad51 into nuclear foci in human glioma cells. The combination of vector-mediated silencing of Rad51 expression and treatment with ionizing radiation resulted in a pronounced reduction of the survival of human glioma cells in culture. In athymyc mice, a single intratumoral injection of Rad51-specific HSV-1 amplicon vector followed by a single radiation treatment resulted in a significant decrease in tumor size. In control animals, including mice that received an intratumoral injection of Rad51-specific amplicon vector but no radiation treatment, the tumor sizes increased.

Search for physical interaction between BICP0 of bovine herpesvirus-1 and p53 tumor suppressor protein.

The immediate-early (IE) protein BICP0 of bovine herpesvirus-1 (BHV-1) may have other functions besides transactivation of viral promoters. Recently, we observed that BICP0, delivered to cultured cells by a helpervirus-free amplicon system, forms spherical or doughnut-like structures in which the tumor suppressor protein p53 is sequestered. The objective was to determine whether BICP0 and p53 interact physically, we used both yeast and mammalian two-hybrid systems. As a bait plasmid, pVA3 which encodes a hybrid protein consisting of the Gal4 DNA binding domain fused to murine p53 was used. The BICP0 gene or its truncated versions were inserted into the prey plasmid pGAD424. Bait and prey plasmids were cotransformed into yeast strain Y153, which has LacZ and HIS3 reporter genes under the control of Gal4 upstream activating sequence. After 4-6 days, colonies were stained for beta-galactosidase activity. In the mammalian two-hybrid system, pM-53 was used as a bait where truncated p53 fused to Gal4 DNA binding domain is expressed. The BICP0 gene was cloned into prey plasmid pVP16. The interaction between p53 and SV40 T-antigen was evaluated as a positive control in both systems. Neither full-length BICP0 nor its truncated derivatives induced beta-galactosidase activity in yeast whereas the positive control turned blue under the same conditions. The mammalian two-hybrid system, in which chloramphenicol acetyltransferase (CAT) activity was used as a reporter, also failed to show an interaction between these two proteins. Co-localization of p53 with BICP0 in spherical structures is unlikely to result from a direct physical interaction between these two proteins. Mediation by additional cellular proteins may be required.

Cytoprotective effect of trimetazidine on 75 min warm renal ischemia-reperfusion injury in rats.

In this experimental study, we evaluated the effect of trimetazidine (TMZ) on renal ischemia-reperfusion (IR) injury in Sprague-Dawley rats. Renal IR was achieved by a 75-min clamping of the left renal pedicle and subsequent 24 h reperfusion, after right nephrectomy was performed. The rats were randomly divided into three groups: group 1 (sham-operated: no IR injury), group 2 (ischemic control: saline treatment), and group 3 (3 mg/kg TMZ before ischemia). After 24 h of reperfusion, blood samples and renal tissue samples were taken to measure the levels of creatinine, tissue malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) activity. Histopathological changes were evaluated. In addition, the 7-day survival rates in each group were evaluated. We found significant increases in the levels of creatinine and tissue MDA, severe acute tubular necrosis, and a significant decrease in the activity of the GSH-Px in group 2. There were significant decreases in the levels of creatinine and tissue MDA, mild acute tubular necrosis, and a significant increase in activity of the GSH-Px in group 3 when compared with the control group (p <0.05). Statistically significant differences (p <0.05) in survival were noted between the ischemic control and sham-operated and TMZ groups. We have concluded that TMZ is able to protect the kidney from warm IR injury.

Determination of glutathione, glutathione reductase, glutathione peroxidase and glutathione S-transferase levels in human lung cancer tissues.

This study was performed to elucidate the lung glutathione-related defense potential in tumoral tissues. Reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase (GR), selenium-dependent (SeGPx) and total glutathione peroxidase (tGPx), and glutathione S-transferase (GST) activities in 38 tumoral lung tissues and 17 normal lung tissues were determined to obtain a comprehensive profile of the lung glutathione and glutathione-related enzymes in cancer. The enzyme levels in tumoral tissues (n = 38) were found to be significantly higher (P < 0.05) than those in normal tissues (n = 17). Reduced glutathione levels, and not oxidized glutathione levels, were found to be higher in normal tissues than those in tumoral tissues. We found no statistically significant difference between the adenocarcinoma and squamous cell carcinoma groups for any of the parameters studied.