Current Molecular Medicine - Volume 17, Issue 3, 2017

Common cerebral small vessel disease (cSVD) abnormalities are a common neuroradiological finding, especially in the elderly. They are associated with a wide clinical spectrum that leads to an increasing disability, impaired global function outcome and a reduced quality of life. A strong association is demonstrated with age and hypertension and other common vascular risk factors, including diabetes mellitus, dyslipoproteinemia, smoking, low vitamin B12 level, and hyperomocysteinemia. Although these epidemiological associations suggest a systemic involvement, etiopathogenetic mechanisms remain unclear. This review focuses on the potential role of endothelial dysfunction and oxidative stress in the pathogenic cascade leading to cSVD. We stressed on the central role of those pathways, and suggest the importance of quantifying the cerebral (and non-only) “endotheliopathic and oxidative load” and its clinical presentation that could lead to a better determination of vascular risk degree. In addition, understanding underlying pathogenic mechanisms could allow us to slow down the progression of vascular damage and, therefore, prevent the disability due to reiterated microvascular damage.

Anaplastic thyroid carcinoma is the rarest but extremely aggressive thyroid cancer subtype. This neoplasia is composed of undifferentiated tumor cells with poor prognosis and resistant to common thyroid cancer therapy. Early stage identification of this cancer for prompt treatment is very vital. Presently, cytological evaluation of fine needle aspiration biopsy (FNAB) which is known as invasive recognition assay, is the standard diagnostic method for the diagnosis of malignant thyroid tumors. Frequent studies have suggested that using the molecular biomarkers of thyroid cancer tissue alongside cytological examination, increase the accuracy of diagnostic tests. Also, these agents could be beneficial for effective target therapy and personalize medicine. In this review, the molecular biomarkers that are involved in anaplastic thyroid carcinoma in four category (gene mutation profile, epigenetic profile, microRNA profile and cancer stem cell markers) were summarized.

Background: Proteotoxic stress and transforming growth factor (TGFβ)-induced epithelial-mesenchymal transition (EMT) are two main contributors of intraocular fibrotic disorders, including proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). However, how these two factors communicate with each other is not well-characterized. Objective: The aim was to investigate the regulatory role of proteotoxic stress on TGFβ signaling in retinal pigment epithelium. Methods: ARPE-19 cells and primary human retinal pigment epithelial (RPE) cells were treated with proteasome inhibitor MG132 and TGFβ. Cell proliferation was analyzed by CCK-8 assay. The levels of mesenchymal markers α-SMA, fibronectin, and vimentin were analyzed by real-time polymerase chain reaction (PCR), western blot, and immunofluorescence. Cell migration was analyzed by scratch wound assay. The levels of p-Smad2, total Smad2, p-extracellular signal-regulated kinase 1/2 (ERK1/2), total ERK1/2, p-focal adhesion kinase (FAK), and total FAK were analyzed by western blot. The mRNA and protein levels of TGFβ receptor-II (TGFβR-II) were measured by realtime PCR and western blot, respectively. Results: MG132-induced proteotoxic stress resulted in reduced cell proliferation. MG132 significantly suppressed TGFβ-induced upregulation of α-SMA, fibronectin, and vimentin, as well as TGFβ-induced cell migration. The phosphorylation levels of Smad2, ERK1/2, and FAK were also suppressed by MG132. Additionally, the mRNA level and protein level of TGFβR-II decreased upon MG132 treatment. Conclusion: Proteotoxic stress suppressed TGFβ-induced EMT through downregulation of TGFβR-II and subsequent blockade of Smad2, ERK1/2, and FAK activation.

Background: Breast cancer metastasis is a highly prevalent cause of death for European females. DNA microarray analysis has established that primary tumors, which remain localized, differ in gene expression from those that metastasize. Crossanalysis of these studies allow to revile the differences that may be used as predictive in the disease prognosis and therapy. Objective: The aim of the project was to validate suggested prognostic and therapeutic markers using meta-analysis of data on gene expression in metastatic and primary breast cancer tumors. Method: Data on relative gene expression values from 12 studies on primary breast cancer and breast cancer metastasis were retrieved from Genevestigator (Nebion) database. The results of the data meta-analysis were compared with results of literature mining for suggested metastatic breast cancer markers and vectors and consistency of their reported differential expression. Results: Our analysis suggested that transcriptional expression of the COX2 gene is significantly downregulated in metastatic tissue compared to normal breast tissue, but is not downregulated in primary tumors compared with normal breast tissue and may be used as a differential marker in metastatic breast cancer diagnostics. RRM2 gene expression decreases in metastases when compared to primary breast cancer and could be suggested as a marker to trace breast cancer evolution. Our study also supports MMP1, VCAM1, FZD3, VEGFC, FOXM1 and MUC1 as breast cancer onset markers, as these genes demonstrate significant differential expression in breast neoplasms compared with normal breast tissue. Conclusion: COX2 and RRM2 are suggested to be prominent markers for breast cancer metastasis. The crosstalk between upstream regulators of genes differentially expressed in primary breast tumors and metastasis also suggests pathways involving p53, ER1, ERB-B2, TNF and WNT, as the most promising regulators that may be considered for new complex drug therapeutic interventions in breast cancer metastatic progression.

Purpose: Corticosteroids remain the mainstay therapy for uveitis, a major cause of blindness in the working age population. However, a substantial number of patients cannot benefit from the therapy due to steroids resistance or intolerance. Tacrolimus has been used to treat refractory uveitis through systemic administration. The aim of this study was to evaluate the therapeutic potential of 0.03% tacrolimus eyedrop in mouse models of uveitis. Methods: 0.03% tacrolimus in perfluorobutylpentane (F4H5) (0.03% Tacrolimus/SFA) was formulated using a previously published protocol. Tacrolimus suspended in PBS (0.03% Tacrolimus/PBS) was used as a control. In addition, 0.1% dexamethasone (0.1% DXM) was used as a standard therapy control. Endotoxin-induced uveitis (EIU) and experimental autoimmune uveoretinitis (EAU) were induced in adult C57BL/6 mice using protocols described previously. Mice were treated with eyedrops three times/day immediately after EIU induction for 48 h or from day 14 to day 25 post-immunization (for EAU). Clinical and histological examinations were conducted at the end of the experiment. Pharmacokinetics study was conducted in mice with and without EIU. At different times after eyedrop treatment, ocular tissues were collected for tacrolimus measurement. Results: The 0.03% Tacrolimus/SFA eyedrop treatment reduced the clinical scores and histological scores of intraocular inflammation in both EIU and EAU to the levels similar to 0.1% DXM eyedrop treatment. The 0.03% Tacrolimus/PBS did not show any suppressive effect in EIU and EAU. Pharmacokinetic studies showed that 15 min after topical administration of 0.03% Tacrolimus/SFA, low levels of tacrolimus were detected in the retina (48 ng/g tissue) and vitreous (2.5 ng/ml) in normal mouse eyes, and the levels were significantly higher in EIU eyes (102 ng/g tissue in the retina and 24 ng/ml in the vitreous). Tacrolimus remained detectable in intraocular tissues of EIU eyes 6 h after topical administration (68 ng/g retinal tissue, 10 ng/ml vitreous). Only background levels of tacrolimus were detected in the retina (2-8 ng/g tissue) after 0.03% Tacrolimus/PBS eyedrop administration. Conclusion: 0.03% Tacrolimus/SFA eyedrop can penetrate ocular barrier and reach intraocular tissue at therapeutic levels in mouse eyes, particularly under inflammatory conditions. 0.03% Tacrolimus/SFA eyedrop may have therapeutic potentials for inflammatory eye diseases including uveitis.

Background: Arrestin domain-containing protein 3 (ARRDC3) is a member of the mammalian α-arrestins family, which has been identified as a tumor suppressor gene in human breast cancer, but its functions are still not clear in human prostate cancer (PCa). Objective: The purpose of the present study was to investigate clinical significance, biological functions and underlying mechanisms of ARRDC3 deregulation in PCa. Method: Involvement of ARRDC3 deregulation in malignant phenotypes of PCa was demonstrated by clinical sample evaluation, microarray analysis, and in vitro and in vivo experiments. The mechanisms underlying its regulatory effect on tumor progression were determined. Results: Microarray analysis found that ARRDC3 low expression was significantly associated with high Gleason score in TMA, and the expression level of ARRDC3 was negatively correlated with Gleason score, metastasis and biochemical recurrence in online Taylor Dataset. As revealed by the dataset, Kaplan-Meier analyses revealed that the biochemical recurrence-free survival (BCR-free) time of PCa patients with ARRDC3 high expression was longer than those with ARRDC3 low expression. Additionally, both univariate and multivariate analyses showed that the downregulation of ARRDC3 was an independent prognostic marker for BCR-free survival of patients with PCa. In vitro studies revealed that ARRDC3 could inhibit proliferation, migration and invasion of PCa cell lines. In vivo studies proved that ARRDC3 over-expressing cells formed significantly larger tumor nodules and remarkably speeded up tumor xenografts growth compared with the controls. Moreover, immunohistochemical scores of Ki67 and MMP-9 were significantly lower than those of the control group. Finally, correlation analysis indicated that the expression of ARRDC3 was negatively correlated with ITGβ ;4 in clinical PCa tissues and cell lines. Conclusion: Our data revealed that ARRDC3 can serve as a tumor suppressor to inhibit PCa progression and an independent marker to predict the risk of biochemical recurrence and metastasis after radical resection of PCa.

Background: An alternative treatment for growth hormone deficiency based on hGH-DNA administration, followed by electro gene transfer, was investigated by injecting the plasmid into surgically exposed or non-exposed quadriceps or tibialis muscle of immunodeficient “little” mice. Methods: An optimization of electrotransfer conditions via a new combination of high/low voltage pulses is presented. After 3 days, serum hGH was determined and in a 28-day assay, the relative growth parameters were compared. Results: Both groups exhibited similar results: 5.0 ± 2.2 (SD) and 3.5 ± 0.9 ng hGH/ml (P>0.05; n=7) for the exposed quadriceps and non-exposed tibialis treatments, respectively. The final body weight increases were 16.1% for the quadriceps and 18.9% for the tibialis group. The tail and nose-to-tail length increases were 4.5% and 7.1% for the quadriceps and 4.8 and 4.6% for the tibialis group. The right and left femur length increases, obtained from radiographic measurements, were 16.9% and 12.7% for the quadriceps and 19.4% and 12.3% for the tibialis, respectively. A non-significant difference between exposed quadriceps and non-exposed tibialis treatments (P=0.48) was confirmed via a completely integrated statistical analysis. Circulating mIGF-1 levels were 126 ± 47, 106 ± 93 (P>0.05) and 38 ± 15 ng/ml for the quadriceps, tibialis and saline treatments, respectively. Conclusion: These results show that hGH-DNA administration into non-exposed tibialis muscle followed by the new HV/LV electrotransfer protocol was an equally efficient, less traumatic treatment, much more suitable for pre-clinical testing than administration into exposed quadriceps.

Background: L-Tetrahydropalmatine (L-THP) is a tetra-hydro protoberberine isoquinoline alkaloid. The phyto-compounds bearing isoquinoline alkaloids have been reported to show a potential effect against a number of human cancers cell lines including leukemia. We hypothesized that L-THP, being an isoquinoline alkaloid, could be a potential molecule against acute lymphoblastic leukemia (ALL), in this study, we evaluate L-THP against p53 deficient leukemia EU-4 cell lines in vitro. Methods: For the study, p53 null leukemia EU-4 cells were used and treated with LTHP. The extent of apoptosis and viability of cells were determined. Expression of apoptosis related proteins such as XIAP and MDM2 was done by western blot and PCR studies. The expression of MDM2 and XIAP was knocked down by small interfering RNA (siRNA). Results: Outcomes of the study suggested that L-THP caused p53-indipendent apoptosis mediated by XIAP in EU-4 cells. The treatment of L-THP caused a decrease in the levels of XIAP protein with increasing dose and time. L-THP caused down-regulation of XIAP protein via inhibiting the expression of MDM2 and involving proteasomedependent pathway. Also, the outcomes of experiments suggested increased sensitivity of leukemia cells towards doxorubicin due to the inhibition of XIAP by L-THP or by siRNA. Conclusion: Findings of the study confirm that L-THP resulted in p53 independent apoptosis via down-regulating XIAP protein by inhibiting MDM2 associated with proteasome-dependent pathway and increased sensitivity of EU-4 cells against doxorubicin. L-THP caused activation of caspase and resulted in apoptosis, L-THP may be a novel molecule for inducing apoptosis specifically in p53 null leukemia EU-4 cells.