The small molecules, natural antioxidant Caffeic Acid (trans-3,4-Dihydroxycinnamic acid CA) and anti-diabetic drug Metformin (Met), activate 5′-adenosine monophosphate-activated protein kinase (AMPK) and interfere with metabolic reprogramming in human cervical squamous carcinoma cells. Here, to gain more insight into the ability of CA, Met and the combination of both compounds to impair aerobic glycolysis (the “Warburg effect”) and disrupt bioenergetics of cancer cells, we employed the cervical tumor cell lines C-4I and HTB-35/SiHa. In epithelial C-4I cells derived from solid tumors, CA alleviated glutamine anaplerosis by downregulation of Glutaminase (GLS) and Malic Enzyme 1 (ME1), which resulted in the reduction of NADPH levels. CA treatment of the cells altered tricarboxylic acid (TCA) cycle supplementation with pyruvate via Pyruvate Dehydrogenase Complex (PDH), increased ROS formation and enhanced cell death. Additionally, CA and CA/Met evoked intracellular energetic stress, which was followed by activation of AMPK and the impairment of unsaturated FA de novo synthesis. In invasive HTB-35 cells, Met inhibited Hypoxia-inducible Factor 1 (HIF-1α) and suppressed the expression of the proteins involved in the “Warburg effect”, such as glucose transporters (GLUT1, GLUT3) and regulatory enzymes of glycolytic pathway Hexokinase 2 (HK2), 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 4 (PFKFB4), Pyruvate Kinase (PKM) and Lactate Dehydrogenase A (LDH). Met suppressed the expression of c-Myc, BAX and cyclin-D1 (CCND1) and evoked apoptosis in HTB-35 cells. In conclusion, both small molecules CA and Met are capable of disrupting energy homeostasis, regulating oxidative metabolism/glycolysis in cervical tumor cells in regard to specific metabolic phenotype of the cells. CA and Met may provide a promising approach in the prevention of cervical cancer progression.

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

The severity of Duchenne muscular dystrophy (DMD), an incurable disease caused by the lack of dystrophin, might be modulated by different factors, including miRNAs. Among them, miR-378 is considered of high importance for muscle biology, but intriguingly, its role in DMD and its murine model ( mdx mice) has not been thoroughly addressed so far. Here, we demonstrate that dystrophic mice additionally globally lacking miR-378 (double-KO [dKO] animals) exhibited better physical performance and improved absolute muscle force compared with mdx mice. Accordingly, markers of muscle damage in serum were significantly decreased in dKO mice, accompanied by diminished inflammation, fibrosis, and reduced abundance of regenerating fibers within muscles. The lack of miR-378 also normalized the aggravated fusion of dystrophin-deficient muscle satellite cells (mSCs). RNA sequencing of gastrocnemius muscle transcriptome revealed fibroblast growth factor 1 ( Fgf1 ) as one of the most significantly downregulated genes in mice devoid of miR-378, indicating FGF1 as one of the mediators of changes driven by the lack of miR-378. In conclusion, we suggest that targeting miR-378 has the potential to ameliorate DMD pathology.

Aims: MicroRNA-378a, highly expressed in skeletal muscles, was demonstrated to affect myoblasts differentiation and to promote tumour angiogenesis. We hypothesized that miR-378a could play a pro-angiogenic role in skeletal muscle and may be involved in regeneration after ischaemic injury in mice. Methods and results: Silencing of miR-378a in murine C2C12 myoblasts did not affect differentiation but impaired their secretory angiogenic potential towards endothelial cells. miR-378a knockout (miR-378a−/−) in mice resulted in a decreased number of CD31-positive blood vessels and arterioles in gastrocnemius muscle. In addition, diminished endothelial sprouting from miR-378a−/− aortic rings was shown. Interestingly, although fibroblast growth factor 1 (Fgf1) expression was decreased in miR-378a−/− muscles, this growth factor did not mediate the angiogenic effects exerted by miR-378a. In vivo, miR-378a knockout did not affect the revascularization of the ischaemic muscles in both normo- and hyperglycaemic mice subjected to femoral artery ligation (FAL). No difference in regenerating muscle fibres was detected between miR-378a−/− and miR-378+/+ mice. miR-378a expression temporarily declined in ischaemic skeletal muscles of miR-378+/+ mice already on Day 3 after FAL. At the same time, in the plasma, the level of miR-378a-3p was enhanced. Similar elevation of miR-378a-3p was reported in the plasma of patients with intermittent claudication in comparison to healthy donors. Local adeno-associated viral vectors-based miR-378a overexpression was enough to improve the revascularization of the ischaemic limb of wild-type mice on Day 7 after FAL, what was not reported after systemic delivery of vectors. In addition, the number of infiltrating CD45+ cells and macrophages (CD45+ CD11b+ F4/80+ Ly6G−) was higher in the ischaemic muscles of miR-378a−/− mice, suggesting an anti-inflammatory action of miR-378a. Conclusions: Data indicate miR-378a role in the pro-angiogenic effect of myoblasts and vascularization of skeletal muscle. After the ischaemic insult, the anti-angiogenic effect of miR-378a deficiency might be compensated by enhanced inflammation.