Rho GTPase activating protein 21-mediated regulation of prostate cancer associated 3 gene in prostate cancer cell.

The overexpression of the prostate cancer antigen 3 (PCA3) gene is well-defined as a marker for prostate cancer (PCa) diagnosis. Although widely used in clinical research, PCA3 molecular mechanisms remain unknown. Herein we used phage display technology to identify putative molecules that bind to the promoter region of PCA3 gene and regulate its expression. The most frequent peptide PCA3p1 (80%) was similar to the Rho GTPase activating protein 21 (ARHGAP21) and its binding affinity was confirmed using Phage Bead ELISA. We showed that ARHGAP21 silencing in LNCaP prostate cancer cells decreased PCA3 and androgen receptor (AR) transcriptional levels and increased prune homolog 2 (PRUNE2) coding gene expression, indicating effective involvement of ARHGAP21 in androgen-dependent tumor pathway. Chromatin immunoprecipitation assay confirmed the interaction between PCA3 promoter region and ARHGAP21. This is the first study that described the role of ARHGAP21 in regulating the PCA3 gene under the androgenic pathway, standing out as a new mechanism of gene regulatory control during prostatic oncogenesis.

An early onset benign myopathy with glycogen storage caused by a de novo 1.4 Mb-deletion of chromosome 14.

Early onset myopathies are a clinically and histologically heterogeneous monogenic diseases linked to approximately 90 genes. Molecular diagnosis is challenging, especially in patients with a mild phenotype. We describe a 26-year-old man with neonatal hypotonia, motor delay and seizures during infancy, and non-progressive, mild muscular weakness in adulthood. Serum Creatine kinase level was normal. Whole-body muscle MRI showed thin muscles, and brain MRI was unremarkable. A deltoid muscle biopsy showed glycogen storage. WGS revealed a de novo 1.4 Mb-deletion of chromosome 14, confirmed by Array-CGH. This microdeletion causes the loss of ten genes including RALGAPA1, encoding for RalA, a regulator of glucose transporter 4 (GLUT4) expression at the membrane of myofibers. GLUT4 was overexpressed in patient's muscle. Here we highlight the importance to search for chromosomal alterations in the diagnostic workup of early onset myopathies.

Whole-Exome Sequencing Indicated New Candidate Genes Associated with Unilateral Cryptorchidism in Pigs.

INTRODUCTION: Cryptorchidism is a hereditary anomaly characterized by the incomplete descent of one or both testicles to the scrotum. One of the challenges of this anomaly is that the retained testicle maintains its endocrine function. As a consequence, cryptorchid animals produce hormone-tainted meat in comparison to castrated animals and are likely to be more aggressive. Cryptorchidism can lead to reduced animal welfare outcomes and cause economic losses. Identifying genetic markers for cryptorchidism is an essential step toward mitigating these negative outcomes and may facilitate genome manipulation to reduce the occurrence of cryptorchidism. Attempts to identify such markers have used genome-wide association studies. Using whole-exome sequencing, we aimed to identify single nucleotide polymorphisms (SNPs) in the coding regions of cryptorchid pigs and to characterize functional pathways concerning these SNPs. METHODS: DNA was extracted and sequenced from 5 healthy and 5 cryptorchid animals from the Landrace breed, using the Illumina HiSeq 2500 platform. Data were pre-processed using the SeqyClean tool and further mapped against the swine reference genome (Sus scrofa 11.1) using BWA software. GATK was used to identify polymorphisms (SNPs and InDels), which were annotated using the VEP tool. Network prediction and gene ontology enrichment analysis were conducted using the Cytoscape platform, and STRING software was used for visualization. RESULTS: A total of 63 SNPs were identified across the genes PIGB, CCPG1, COMMD9, LDLRAD3, TRIM44, MYLPF, SEPTIN, ZNF48, TIA1, FAIM2, KRT18, FBP1, FBP2, CTSL, DAPK1, DHX8, GPR179, DEPDC1B, ENSSSCG00000049573, ENSSSCG00000016384, ENSSSCG00000022657, ENSSSCG00000038825, and ENSSSCG00000001229. Using pathway enrichment analyses and network prospection, we have identified the following significant adjusted p value threshold of 0.001 involved with the biological function pathways of estrogen signaling, cytoskeleton organization, and the pentose phosphate pathway. CONCLUSION: Our data suggest the involvement of new SNPs and genes in developing cryptorchidism in pigs. However, further studies are needed to validate our results in a larger cohort population. Variations in the GPR179 gene, with implications at the protein level, may be associated with the appearance of this anomaly in the swine. Finally, we are showing that the estrogen signaling pathway may be involved in the pathophysiological mechanisms of this congenital anomaly as previously reported in GWAS.

Genetic polymorphisms and their effects on the severity of silicosis in workers exposed to silica in Brazil.

OBJECTIVE: Silicosis is a pneumoconiosis characterized by fibrosis of the lung parenchyma caused by inhalation of silica particles. Genetic factors might play a role in the severity silicosis. We sought to evaluate the influence of polymorphisms in the ACE, FAS, FASLG, NOS2, IL1RN, FAM13A, TGFB1, and TNF genes on the severity of silicosis. METHODS: Nine polymorphisms were genotyped by PCR in a sample of 143 patients with silicosis in the state of Rio de Janeiro, Brazil. RESULTS: Fifty-seven patients (40%) were classified as having simple silicosis and 86 (60%) were classified as having complicated silicosis. The TT genotype of rs1800469 in the TGFB1 gene showed a protective effect for complicated silicosis (OR = 0.35; 95% CI, 0.14-0.92; p = 0.028) when compared with the other two genotypes (CC+CT). The polymorphic T allele of rs763110 in the FASLG gene (OR = 0.56; 95% CI, 0.31-0.99; p = 0.047), as well as a dominant model for the T allele (TT+CT: OR = 0.37; 95% CI, 0.15-0.96; p = 0.037), also showed a protective effect. When patients with simple silicosis despite having been exposed to silica for a longer time (> 44,229 hours) were compared with patients with complicated silicosis despite having been exposed to silica for a shorter time, the T allele of rs763110 in the FASLG gene (OR = 0.20; 95% CI, 0.08-0.48; p < 0.0001), as well as dominant and recessive models (OR = 0.06; 95% CI, 0.00-0.49; p = 0.01 and OR = 0.22; 95% CI, 0.06-0.77; p = 0.014, respectively), showed a protective effect against the severity of silicosis. CONCLUSIONS: It appears that rs1800469 polymorphisms in the TGFB1 gene and rs763110 polymorphisms in the FASLG gene are involved in the severity of silicosis. Given the lack of studies relating genetic polymorphisms to the severity of silicosis, these results should be replicated in other populations.

Transporters, TBC1D4, and ARID5B Variants to Explain Glycated Hemoglobin Variability in Patients with Type 2 Diabetes.

INTRODUCTION: Genetic variants could aid in predicting antidiabetic drug response by associating them with markers of glucose control, such as glycated hemoglobin (HbA1c). However, pharmacogenetic implementation for antidiabetics is still under development, as the list of actionable markers is being populated and validated. This study explores potential associations between genetic variants and plasma levels of HbA1c in 100 patients under treatment with metformin. METHODS: HbA1c was measured in a clinical chemistry analyzer (Roche), genotyping was performed in an Illumina-GSA array and data were analyzed using PLINK. Association and prediction models were developed using R and a 10-fold cross-validation approach. RESULTS: We identified genetic variants on SLC47A1, SLC28A1, ABCG2, TBC1D4, and ARID5B that can explain up to 55% of the interindividual variability of HbA1c plasma levels in diabetic patients under treatment. Variants on SLC47A1, SLC28A1, and ABCG2 likely impact the pharmacokinetics (PK) of metformin, while the role of the two latter can be related to insulin resistance and regulation of adipogenesis. CONCLUSIONS: Our results confirm previous genetic associations and point to previously unassociated gene variants for metformin PK and glucose control.

Increased glucose metabolism in Arid5b-/- skeletal muscle is associated with the down-regulation of TBC1 domain family member 1 (TBC1D1).

BACKGROUND: Skeletal muscle has an important role in regulating whole-body energy homeostasis, and energy production depends on the efficient function of mitochondria. We demonstrated previously that AT-rich interactive domain 5b (Arid5b) knockout (Arid5b-/-) mice were lean and resistant to high-fat diet (HFD)-induced obesity. While a potential role of Arid5b in energy metabolism has been suggested in adipocytes and hepatocytes, the role of Arid5b in skeletal muscle metabolism has not been studied. Therefore, we investigated whether energy metabolism is altered in Arid5b-/- skeletal muscle. RESULTS: Arid5b-/- skeletal muscles showed increased basal glucose uptake, glycogen content, glucose oxidation and ATP content. Additionally, glucose clearance and oxygen consumption were upregulated in Arid5b-/- mice. The expression of glucose transporter 1 (GLUT1) and 4 (GLUT4) in the gastrocnemius (GC) muscle remained unchanged. Intriguingly, the expression of TBC domain family member 1 (TBC1D1), which negatively regulates GLUT4 translocation to the plasma membrane, was suppressed in Arid5b-/- skeletal muscle. Coimmunofluorescence staining of the GC muscle sections for GLUT4 and dystrophin revealed increased GLUT4 localization at the plasma membrane in Arid5b-/- muscle. CONCLUSIONS: The current study showed that the knockout of Arid5b enhanced glucose metabolism through the downregulation of TBC1D1 and increased GLUT4 membrane translocation in skeletal muscle.

RASAL1 induces to downregulate the SCD1, leading to suppression of cell proliferation in colon cancer via LXRα/SREBP1c pathway.

BACKGROUND: Recent studies have confirmed that RASAL1 has an antitumor effect in many cancers, but its functional role and the molecular mechanism underlying in colon cancer has not been investigated. RESULTS: We collected human colon cancer tissues and adjacent non-tumor tissues, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and normal colonic mucosa cell line NCM460. RT-qPCR was used to detect the RASAL1 level in the clinical tissues and cell lines. In LoVo and HCT-116, RASAL1 was artificially overexpressed. Cell viability and proliferation were measured using CCK-8 assays, and cell cycle was detected via PI staining and flow cytometry analysis. RASAL1 significantly inhibited the cell proliferation via inducing cell cycle arrest, suppressed cell cycle associated protein expression, and decreased the lipid content and inhibited the SCD1 expression. Moreover, SCD1 overexpression induced and downregulation repressed cell proliferation by causing cell cycle arrest. Additionally, luciferase reporter assays were performed to confirm the direct binding between SREBP1c, LXRα and SCD1 promoter, we also demonstrated that RASAL1 inhibit SCD1 3'-UTR activity. RASAL1 inhibited tumor growth in xenograft nude mice models and shows inhibitory effect of SCD1 expression in vivo. CONCLUSION: Taken together, we concluded that RASAL1 inhibited colon cancer cell proliferation via modulating SCD1 activity through LXRα/SREBP1c pathway.

PINK1 Silencing Modifies Dendritic Spine Dynamics of Mouse Hippocampal Neurons.

PTEN-induced kinase 1 (PINK1) mutations can cause early-onset Parkinson's disease and patients are likely to develop cognitive decline, depression, and dementia. Several neurophysiological studies have demonstrated PINK1 deficiency impairs striatal and hippocampal presynaptic plasticity. Dendritic spine postsynaptic abnormalities are common in neurological diseases; however, whether PINK1 silencing modifies dendritic spine dynamics of hippocampal neurons is unclear. To address this question, confocal images of mouse cultured hippocampal neurons transfected with plasmids to silence PINK1 were analyzed. These studies revealed that PINK1 silencing increased density of thin spines and reduced head size of stubby spines. Immunoblotting analysis uncovered that PINK1 silencing decreased expression of postsynaptic density proteins (PSD95 and Shank) and glutamate receptors (NR2B and mGluR5). We also found PINK1 silencing regulated dendritic spine morphology by actin regulatory proteins (RhoGAP29 and ROCK2) and regulated neuronal survival by decreased Akt activation. These results suggest PINK1 may regulate postsynaptic plasticity in hippocampal neurons generating presymptomatic alterations in dendritic spines that eventually could lead to the neurodegeneration and cognitive decline often seen in Parkinson's disease.

The RabGAP Gene Family in Tomato (Solanum lycopersicum) and Wild Relatives: Identification, Interaction Networks, and Transcriptional Analysis during Plant Development and in Response to Salt Stress.

RabGTPase activating proteins (RabGAP) are responsible for directing the deactivation of vesicular trafficking master regulators associated to plant development, the RabGTPase proteins. Recently, RabGAPs were identified in Arabidopsis and rice, but studies were not yet reported in tomato. Herein, we identified 24 RabGAP-encoding genes in cultivated tomato (Solanum lycopersicum) and its wild relative genomes (Solanum pimpinellifolium and Solanum pennellii). We analyzed them based on their exon-intron structures, conserved protein motifs, putative subcellular localizations, phylogenetic and gene duplications analyses, interaction networks, and gene expression patterns in tomato. Phylogenetic relationship analysis also indicated that RabGAP family is classified into seven subclasses, of which subclasses I and II are plant-exclusive. Furthermore, segmental duplication events and positive evolutionary forces are associated with the maintenance of the number and function of their members. On the other hand, the protein-protein interaction networks on tomato suggested that members of subclasses I, II, and III could be associated to endocytic traffic routes. In addition, the qRT-PCR experiments in S. lycopersicum and Solanum chilense exposed to a salt stress treatment validated the differential expression patterns of 20 RabGAP genes in different tissues, development stages, and stress conditions obtained through extensive microarray-based analyses. This work suggests the critical role of RabGAP family in the context of intracellular vesicular trafficking in tomato, particularly under conditions of abiotic stress. It also contributes to the breeding programs associated with the development of crops tolerant to salt stress.

ARHGAP21 deficiency impairs hepatic lipid metabolism and improves insulin signaling in lean and obese mice.

ARHGAP21 is a Rho-GAP that controls GTPases activity in several tissues, but its role on liver lipid metabolism is unknown. Thus, to achieve the Rho-GAP role in the liver, control and ARHGAP21-haplodeficient mice were fed chow (Ctl and Het) or high-fat diet (Ctl-HFD and Het-HFD) for 12 weeks, and pyruvate and insulin tolerance tests, insulin signaling, liver glycogen and triglycerides content, gene and protein expression, and very-low-density lipoprotein secretion were measured. Het mice displayed reduced body weight and plasma triglycerides levels, and increased liver insulin signaling. Reduced gluconeogenesis and increased glycogen content were observed in Het-HFD mice. Gene and protein expression of microsomal triglyceride transfer protein were reduced in both Het mice, while the lipogenic genes SREBP-1c and ACC were increased. ARHGAP21 knockdown resulted in hepatic steatosis through increased hepatic lipogenesis activity coupled with decreases in CPT1a expression and very-low-density lipoprotein export. In conclusion, liver of ARHGAP21-haplodeficient mice are more insulin sensitive, associated with higher lipid synthesis and lower lipid export.

Identification and Characterization of Novel Fusion Genes with Potential Clinical Applications in Mexican Children with Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia is the most common type of childhood cancer worldwide. Mexico City has one of the highest incidences and mortality rates of this cancer. It has previously been recognized that chromosomal translocations are important in cancer etiology. Specific fusion genes have been considered as important treatment targets in childhood acute lymphoblastic leukemia (ALL). The present research aimed at the identification and characterization of novel fusion genes with potential clinical implications in Mexican children with acute lymphoblastic leukemia. The RNA-sequencing approach was used. Four fusion genes not previously reported were identified: CREBBP-SRGAP2B, DNAH14-IKZF1, ETV6-SNUPN, ETV6-NUFIP1. Although a fusion gene is not sufficient to cause leukemia, it could be involved in the pathogenesis of the disease. Notably, these new translocations were found in genes encoding for hematopoietic transcription factors which are known to play an important role in leukemogenesis and disease prognosis such as IKZF1, CREBBP, and ETV6. In addition, they may have an impact on the prognosis of Mexican pediatric patients with ALL, with the potential to be included in the current risk stratification schemes or used as therapeutic targets.

RASAL1 induces to downregulate the SCD1, leading to suppression of cell proliferation in colon cancer via LXRα/SREBP1c pathway

BACKGROUND: Recent studies have confirmed that RASAL1 has an antitumor effect in many cancers, but its functional role and the molecular mechanism underlying in colon cancer has not been investigated. RESULTS: We collected human colon cancer tissues and adjacent non-tumor tissues, human colon cancer cell lines LoVo, CaCo2, SW1116, SW480 and HCT-116, and normal colonic mucosa cell line NCM460. RT-qPCR was used to detect the RASAL1 level in the clinical tissues and cell lines. In LoVo and HCT-116, RASAL1 was artificially overexpressed. Cell viability and proliferation were measured using CCK-8 assays, and cell cycle was detected via PI staining and flow cytometry analysis. RASAL1 significantly inhibited the cell proliferation via inducing cell cycle arrest, suppressed cell cycle associated protein expression, and decreased the lipid content and inhibited the SCD1 expression. Moreover, SCD1 overexpression induced and downregulation repressed cell proliferation by causing cell cycle arrest. Additionally, luciferase reporter assays were performed to confirm the direct binding between SREBP1c, LXRα; and SCD1 promoter, we also demonstrated that RASAL1 inhibit SCD1 3'-UTR activity. RASAL1 inhibited tumor growth in xenograft nude mice models and shows inhibitory effect of SCD1 expression in vivo. CONCLUSION: Taken together, we concluded that RASAL1 inhibited colon cancer cell proliferation via modulating SCD1 activity through LXRα/SREBP1c pathway.

ARHGAP21 as a master regulator of multiple cellular processes.

The cellular cytoskeleton is involved with multiple biological processes and is tightly regulated by multiple proteins and effectors. Among these, the RhoGTPases family is one of the most important players. RhoGTPAses are, in turn, regulated by many other elements. In the past decade, one of those regulators, the RhoGAP Rho GTPase Activating Protein 21 (ARHGAP21), has been overlooked, despite being implied as having an important role on many of those processes. In this paper, we aimed to review the available literature regarding ARHGAP21 to highlight its importance and the mechanisms of action that have been found so far for this still unknown protein involved with cell adhesion, migration, Golgi regulation, cell trafficking, and even insulin secretion.

Dynamic relationship between SIPA1 gene and protein expression and the development of gastric cancer.

Association of signal-induced proliferation-associated 1 (SIPA) gene and protein expression with gastric cancer development was examined. SIPA1 mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and western blot, respectively, in 40 gastric tumor and tumor-adjacent normal tissues. SIPA1, VEGF-A, and FVIII levels in 60 gastric tumor and 40 tumor-adjacent normal tissues were examined by immunohistochemical staining. Correlations between SIPA1, VEGF-A, and microvessel density (MVD) were analyzed. SIPA1 mRNA levels were significantly lower in tumor tissues than in tumor-adjacent normal tissues (P < 0.05). Similarly, protein levels were significantly lower in tumor tissues (0.3043 ± 0.1062) than in tumor-adjacent normal tissues (0.5423 ± 0.0682, P < 0.05). Positive staining rates of SIPA1 (48.3%) and VEGF-A (36.7%) were lower and higher, respectively, in tumor tissues than in tumor-adjacent normal tissues (65.0 and 2.5%, P < 0.05). Positive protein staining rates in tumor tissues correlated with the degree of differentiation, lymph node metastases, and clinical grading (P < 0.05) and not with sex, age, or tumor size (P > 0.05). Significantly higher MVD (57.4 ± 9.3) was observed in tumor tissues displaying positive SIPA1 staining than in tumor-adjacent normal tissues (41.2 ± 5.7, P < 0.05). SIPA1 and VEGF-A expression in tumor tissues were negatively correlated (r = -0.736, P < 0.05). SIPA1 and its protein may play important roles in gastric cancer invasion, metastasis, and biological behavior. Low SIPA1 levels in gastric cancer may accelerate tumor development and progression by promoting VEGF-A expression to increase vascular density.

Impact of rare variants in ARHGAP29 to the etiology of oral clefts: role of loss-of-function vs missense variants.

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a prevalent, complex congenital malformation. Genome-wide association studies (GWAS) on NSCL/P have consistently identified association for the 1p22 region, in which ARHGAP29 has emerged as the main candidate gene. ARHGAP29 re-sequencing studies in NSCL/P patients have identified rare variants; however, their clinical impact is still unclear. In this study we identified 10 rare variants in ARHGAP29, including five missense, one in-frame deletion, and four loss-of-function (LoF) variants, in a cohort of 188 familial NSCL/P cases. A significant mutational burden was found for LoF (Sequence Kernel Association Test, p = 0.0005) but not for missense variants in ARHGAP29, suggesting that only LoF variants contribute to the etiology of NSCL/P. Penetrance was estimated as 59%, indicating that heterozygous LoF variants in ARHGAP29 confer a moderate risk to NSCL/P. The GWAS hits in IRF6 (rs642961) and 1p22 (rs560426 and rs4147811) do not seem to contribute to the penetrance of the phenotype, based on co-segregation analysis. Our data show that rare variants leading to haploinsufficiency of ARHGAP29 represent an important etiological clefting mechanism, and genetic testing for this gene might be taken into consideration in genetic counseling of familial cases.

Benefits of L-alanine or L-arginine supplementation against adiposity and glucose intolerance in monosodium glutamate-induced obesity.

PURPOSE: L-alanine (Ala) and L-arginine (Arg) have been reported to regulate pancreatic ß-cell physiology and to prevent body fat accumulation in diet-induced obesity. Here, we assessed growth and adiposity parameters, glucose tolerance, insulin secretion and the expression of insulin and nutrient-regulated proteins in monosodium glutamate (MSG)-obese mice supplemented with either Ala or Arg. METHODS: Male newborn C57Bl/6 mice received a daily subcutaneous injection of MSG or saline solution (CTL group), during the first 6 days of life. From 30 to 90 days of age, MSG and CTL mice received or not 2.55 % Ala (CAla or MArg groups) or 1.51 % Arg-HCl (CArg or MArg groups) in their drinking water. RESULTS: Adult MSG mice displayed higher adiposity associated with lower phosphorylation of the adipogenic enzyme, ACC, in adipose tissue. Glucose intolerance in MSG mice was linked to lower insulin secretion and to lower expression of IRß in adipose tissue, as well as AS160 phosphorylation in skeletal muscle. Perigonadal fat depots were smaller in Ala and Arg mice, while retroperitoneal fat pads were decreased by Ala supplementation only. Both Ala and Arg improved fed-state glycemia as well as IRß and pAS160 content, but only Ala led to improved glucose tolerance and insulin secretion. Adipostatic signals were increased in MAla mice, as indicated by enhanced AMPK phosphorylation and pACC content in fat depots. CONCLUSIONS: Ala supplementation led to more pronounced metabolic improvements compared to Arg, possibly due to suppression of lipogenesis through activation of the AMPK/ACC pathway.

Effect of Combined Action of Extracellular ATP and Elevated Calcium on Osteogenic Differentiation of Primary Cultures From Rat Calvaria.

The in vitro osteogenic differentiation has been intensively studied. However, it is not yet clear precisely how osteogenesis can be optimized. Changes in extracellular Ca(2+) concentration ([Ca(2+) ]e ), as well as modulation of purinergic receptors play an important role in the regulation of osteoblasts differentiation and bone formation. In this study, we investigated the effects of a combined treatment of ATPγ-S and high [Ca(2+) ]e (5.35 mM) on osteogenic differentiation and function of primary cell cultures from rat calvaria. Our results indicate that ATPγ-S stimulates cell transition from the G0 to S phase of cell cycle, involving the PI3K signaling pathway. Treatment with 10 or 100 µM ATPγ-S and [Ca(2+) ]e (ATP-[Ca(2+) ]e ) for 48 h increases cell number significantly above the control. ATPγ-S treatment in osteogenic medium containing [Ca(2+) ]e stimulates the gene expression of BMP-4, BMP-5, and OPN at 16, 48, and 72 h, respectively, above control. In same conditions, treatment for 6 days with 10 µM UTP or 100 µM UDP significantly increased the ALP activity respect to control. Cells grown in osteogenic medium showed a statistically significant increase in calcium deposits at 15 and 18 days, for 10 µM ATPγ-S treatment, and at 18 and 22 days, for [Ca(2+) ]e treatment, respect to control but ATP-[Ca(2+) ]e treatment shown a significant greater mineralization at 15 days respect to ATPγ-S, and at 18 days respect to both agonists. In conclusion, we demonstrated that an osteogenic medium containing 10 µM ATPγ-S and 5.35 mM [Ca(2+) ]e enhance osteogenesis and mineralization by rat primary calvarial cells cultures. J. Cell. Biochem. 117: 2658-2668, 2016. © 2016 Wiley Periodicals, Inc.

Synergistic effect of apoptosis and necroptosis inhibitors in cisplatin-induced nephrotoxicity.

Necroptosis is a nonapoptotic cell death pathway. We aim to study the effect of necrostatin-1 (a specific necroptosis inhibitor) in cisplatin-induced injury. We analyzed the effect of the combined use of inhibitors of apoptosis (z-vad) and necroptosis (necrostatin-1) in acute kidney injury by cisplatin in human proximal tubule cells. Our results showed moderate effectiveness in cytoprotection after treatment with z-vad. But the concomitant use of inhibitors (z-vad and necrostatin-1) presented synergistic and additive protection. The present study analyzed the caspase-3 activity and we observed a significant decrease in the group treated with z-vad and cisplatin. However we did not observe changes in the group treated with both inhibitors (z-vad and necrostatin-1) and cisplatin. Thus, demonstrating that necroptosis is a caspase-independent mechanism. We also analyzed the effect of necrostatin-1 in vivo model. C57BL/6 mice were treated with cisplatin and/or inhibitors. The concomitant use of inhibitors (z-vad and necrostatin-1) recovered renal function and decreased levels of urinary Ngal. Additionally, we analyzed the expression of RIP-1, a specific marker for necroptosis. In animals treated with cisplatin and z-VAD levels of RIP-1 were higher. This result reinforces that necroptosis occurs only in conditions where apoptosis was blocked. However, the use of both inhibitors (z-vad and necrostatin-1) provided additional protection. In conclusion, our study has a significant potential to show in vitro and in vivo protection obtained by necrostatin-1. Therefore, our results suggest that necroptosis may be an important mechanism of cell death after kidney injury.