Urinary bladder cancer is one of the most fatal and expensive diseases of industrialized world. Despite the strenuous efforts, no seminal advances have been achieved for its clinical management. Given the importance of metabolic reprogramming in cancer cell survival and growth, we have herein employed 3-BrPA, a halogenated derivative of pyruvate and historically considered inhibitor of glycolysis, to eliminate bladder cancer cells with highly oncogenic molecular signatures. Bladder cancer cells were exposed to 3-BrPA in the absence or presence of several specific inhibitors. Cell viability was determined by MTT and flow-cytometry assays; cell death, signaling activity and metabolic integrity by Western blotting and immunofluorescence; mutant-gene profiling by DNA sequencing; and gene expression by RT-sqPCR. 3-BrPA could activate dose-dependent apoptosis (type 1 PCD) and regulated necrosis (type 3 PCD) of T24 (grade III; H-RasG12V; p53ΔY126), but not RT4 (grade I), cells, with PARP, MLKL, Drp1 and Nec-7-targeted components critically orchestrating necrotic death. However, similarly to RIPK1 and CypD, p53 presented with non-essential contribution to 3-BrPA-induced cellular collapse, while reactivation of mutant p53 with PRIMA-1 resulted in strong synergism of the two agents. Given the reduced expression of MPC components (likely imposing mitochondrial dysfunction) in T24 cells, the suppression of constitutive autophagy (required by cells carrying oncogenic Ras; also, type 2 PCD) and derangement of glucose-homeostasis determinants by 3-BrPA critically contribute to drug-directed depletion of ATP cellular stores. This bioenergetic crisis is translated to severe dysregulation of Akt/FoxO/GSK-3, mTOR/S6, AMPK and MAPK (p44/42, p38 and SAPK/JNK) signaling pathways in 3-BrPA-treated T24 cells. Sensitivity to 3-BrPA (and tolerance to glucose deprivation) does not rely on B-RafV600E or K-RasG13D mutant oncogenic proteins, but partly depends on aberrant signaling activities of Akt, MAPK and AMPK kinases. Interestingly, MCT1- and macropinocytosis-mediated influx of 3-BrPA in T24 represents the principal mechanism that regulates cellular responsiveness to the drug. Besides its capacity to affect transcription in gene-dependent manner, 3-BrPA can also induce GLUT4-specific splicing silencing in both sensitive and resistant cells, thus dictating alternative routes of drug trafficking. Altogether, it seems that 3-BrPA represents a promising agent for bladder cancer targeted therapy.

Background: Cilostazol is a drug of choice for the treatment of intermittent claudication that also affects innate and adaptive immune cells. The purpose of our study was the evaluation of cilostazol’s impact on the immune and angiogenic response in murine models of hind limb ischemia. Methods: We used 108 immunodeficient NOD.CB17-Prkdcscid/J mice and 108 wild-type CB17 mice. At day 0 (D0), all animals underwent hind limb ischemia. Half of them in both groups received daily cilostazol starting at D0 and for the next 7 postoperative days, while the rest of them served as controls, receiving vehicle. Interleukin (IL) 2, IL-4, IL-6, IL-10, IL-17A, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) serum concentrations were measured by flow cytometry on postsurgery days D1, D3, D5, and D7. On D7, both groups underwent positron emission tomography scan with ⁶⁸Ga-RGD. Mice were euthanatized and gastrocnemius muscles were obtained for histological evaluation. Results: There was a statistically significant augmentation ( P < .05) in IL-4, IL-10, IL-6, and IFN-γ concentrations in treated CB17 animals, while IL-2 was significantly suppressed. Significant difference was detected between the CiBisch and Bisch groups on D1 and D7 ( P < .05) in CD31 staining. In treated NOD.CB17 animals, TNF-α, IL-6, and IFN-γ presented significant augmentation, while ⁶⁸Ga-NODAGA-RGDfK uptake and CD31 expression were found significantly lower for both legs in comparison to the control. Conclusion: Cilostazol seems to significantly increase angiogenesis in wild-type animals during the first postoperational week. It also influences immune cells, altering the type of immune response by promoting anti-inflammatory cytokine production in wild-type animals, while it helps toward inflammation regression in immunodeficient animals.

Blood donors with beta-thalassemia traits (βThal⁺) have proven to be good “storers”, since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored βThal⁺ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted βThal⁺ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that βThal⁺ RBCs present a marginal trend for higher recovery, regardless of the recipient’s immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored βThal⁺ RBCs. Overall, it seems that the intrinsic physiology of βThal⁺ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

One of the commonly used animal models in fertility, developmental and neurobiological studies is the laboratory rat. The early recognition of rat pregnancy and confirmation of the exact embryonic day are vital. The aim of this study was to investigate the correlation of maternal weight at the time of conception to its increase throughout gestation, aiming to develop a mathematical model, which can be used for the determination of the exact day of pregnancy, set the threshold, and monitor pregnancy from the onset. We studied a total of 173 Wistar rats with a mean body weight of 238.22 ± 34.9 g. After 72 h at the male’s cages, we considered as Day 0 (D0) the day in which a copulatory plug or sperm was found during the vaginal smear examination. After that period the female animals were transferred into their cages, and weight monitoring started 14 days (D14) after D0, until parturition. Based on the statistical analysis, there is a correlation between maternal body weight at D0 and maternal body weight from D14 to D19. Moreover, the average weight gain from D14 to D19 is positively correlated to initial female body weight, while there is no correlation between each pregnant animal’s weight from D14 to D19 and litter size. A mathematical model was developed as a tool for the verification of the day of pregnancy. In conclusion, continuous monitoring of maternal weight after D14 can be a reliable method for the recognition of pregnancy and determination of the exact gestational day.