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A combination of miR501-5p and mTOR as molecular markers for the prognosis of renal carcinomas
No full textIn the last years, there is great impetus to discover new biomarkers in RCC in order to address the patients toward an
appropriate adjuvant therapy. In this regard, miR501-5p that is differentially expressed in RCC and mTOR which in many
tumours correlates with a poor prognosis, could represent possible biomarkers. Therefore, miR501-5p expression and
mTOR activity will be studied in normal and RCC tissues as well as in kidney cell lines depleted or enriched in miR501-5p
sequences.
Cell growth, apoptosis, autophagy and related signaling pathways will be analyzed in kidney cells and tissues expressing
different levels of miR501-5p in order to evaluate the role of miR501-5p in RCC.
High and low miR501-5p expression levels combined with mTOR activity of different RCC tissues will be matched with the
outcome of RCC patients to evaluate their possible prognostic role
Role of calcium in polycystic kidney disease: From signaling to pathology
No full textAutosomal dominant polycystic kidney disease (ADPKD) is the most common inherited monogenic kidney disease. Characterized by the development and growth of cysts that cause progressive kidney enlargement, it ultimately leads to end-stage renal disease. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene, while mutations in the PKD2 gene account for the remaining 15% of cases. The PKD1 gene encodes for polycystin-1 (PC1), a large multi-functional membrane receptor protein able to regulate ion channel complexes, whereas polycystin-2 (PC2), encoded by the PKD2 gene, is an integral membrane protein that functions as a calcium-permeable cation channel, located mainly in the endoplasmic reticulum (ER). In the primary cilia of the epithelial cells, PC1 interacts with PC2 to form a polycystin complex that acts as a mechanosensor, regulating signaling pathways involved in the differentiation of kidney tubular epithelial cells. Despite progress in understanding the function of these proteins, the molecular mechanisms associated with the pathogenesis of ADPKD remain unclear. In this review we discuss how an imbalance between functional PC1 and PC2 proteins may disrupt calcium channel activities in the cilium, plasma membrane and ER, thereby altering intracellular calcium signaling and leading to the aberrant cell proliferation and apoptosis associated with the development and growth of renal cysts. Research in this field could lead to the discovery of new molecules able to rebalance intracellular calcium, thereby normalizing cell proliferation and reducing kidney cyst progression
Mitochondrial dysfunction and death in motor neurons exposed to the glutathione-depleting agent ethacrynic acid
No full textThis study investigated the mechanisms of toxicity of glutathione (GSH) depletion in one cell type, the motor neuron. Ethacrynic acid (EA) (100 μM) was added to immortalized mouse motor neurons (NSC-34) to deplete both cytosolic and mitochondrial glutathione rapidly. This caused a drop in GSH to 25% of the initial level in 1 h and complete loss in 4 h. This effect was accompanied by enhanced generation of reactive oxygen species (ROS) with a peak after 2 h of exposure, and by signs of mitochondrial dysfunction such as a decrease in 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) (30% less after 4 h). The increase in ROS and the MTT reduction were both EA concentration-dependent. Expression of heme oxygenase-1 (HO-1), a marker of oxidative stress, also increased. The mitochondrial damage was monitored by measuring the mitochondrial membrane potential (MMP) from the uptake of rhodamine 123 into mitochondria. MMP dropped (20%) after only 1 h exposure to EA, and slowly continued to decline until 3 h, with a steep drop at 5 h (50% decrease), i.e. after the complete GSH loss. Quantification of DNA fragmentation by the TUNEL technique showed that the proportion of cells with fragmented nuclei rose from 10% after 5 h EA exposure to about 65% at 18 h. These results indicate that EA-induced GSH depletion rapidly impairs the mitochondrial function of motor neurons, and this precedes cell death. This experimental model of oxidative toxicity could be useful to study mechanisms of diseases like spinal cord injury (SCI) and amyotrophic lateral sclerosis (ALS), where motor neurons are the vulnerable population and oxidative stress has a pathogenic role
Hyperforin contributes to the hepatic CYP3A-indicing effect of Hypericum perforatum extract in the mouse
No full textThis study in mice investigated whether hyperforin accounts for the inductive effects on cytochrome P4503A enzymes of St. John’s wort extracts (SJW; Hypericum perforatum), one of the most popular herbal preparations because of its alleged activity in mild to moderate depression. A hydroalcoholic extract containing 4.5% hyperforin was given at a dose of 300 mg/kg, bis in die (b.i.d.), for 4 and 12 days. Hyperforin, its main phloroglucinol component, was given as dicyclohexylammonium (DCHA) salt (18.1 mg/kg, b.i.d.) on the basis of its content in the extract, to ensure comparable exposure to hyperforin. The extract increased hepatic erythromycin-N-demethylase (ERND) activity, which is cytochrome P450 enzyme (CYP) 3A-dependent, about 2.2-fold after 4 days of dosing, with only slightly greater effect after 12 days (2.8 times controls). Hyperforin too increased ERND activity within 4 days, much to the same extent as the extract (1.8 times the activity of controls), suggesting that it behaves qualitatively and quantitatively like the extract as regards induction of CYP3A activity. This effect was confirmed by Western blot analysis of hepatic CYP3A expression. Exposure to hyperforin at the end of the 4-day treatment was still similar to that with SJW extract, although it was variable and lower than after the first dose in both cases, further suggesting that hyperforin plays a key role in CYP3A induction by the SJW extract in the mouse. Standardization of the extracts based on the hyperforin content can be proposed for further evaluation of their potential action on first-pass metabolism and clearance of coadministered CYP3A substrates
MicroRNA501 may affect the aggressiveness of clear cell renal carcinoma
No full textINTRODUCTION & OBJECTIVES: MicroRNAs (miR) are small, noncoding RNAs that regulate gene expression and are involved in different biological processes including differentiation, proliferation and apoptosis. Mutations or altered expression of miRs might cause several
diseases including cancer. Since a variable expression of miR501 not related with the patient age or sex in 63 pairs of normal and clear cell renal carcinoma (ccRCC) tissues has been found, we have analysed the possible function of this miR in ccRCC.
MATERIAL & METHODS:
Analysis of miR501 expression was performed by microarray and real time RT-PCR. miR501 up or downregulation was performed by cell transfection with a specific plasmid expressing miR501 sequences (PL-501) and antagomiR, respectively. Apoptosis was studied through caspase-3 activity and cell cycle analysis. Cell proliferation was evaluated by direct cell count
and with the CellTiter method. Protein levels and kinase activity were calculated by using immunological techniques and cell imaging.
RESULTS: Follow up analysis at least 5 years in 35 ccRCC subjects showed a good prognosis for patients with a lower expression (<1) of miR501 in ccRCC tissues compared with normal renal parenchyma. Conversely, 50% of patients with unchanged or higher levels of miR501 exhibited a poor prognosis with a 25% of deaths. In order to evaluate the role of miR501 in renal cancer, we have modified its expression transfecting kidney carcinoma cells KJ29 with a specific antagomiR and with the PL-501 plasmid. MiR501 downregulation caused a reduction of mTOR activity, the increase of G0/G1 phase of cell cycle and induced apoptosis by enhancing the activity of caspase-3. Activation of apoptosis occurred in a p53-dependent manner without affecting the expression of the mTOR-related MDM2 protein, an inhibitor of p53, which results overexpressed in metastatic kidney carcinoma. On the contrary, miR501 upregulation caused mTOR activation, increased expression of MDM2 and enhanced cell proliferation and survival.
CONCLUSIONS: These findings suggest for the miR501 a role of kingmaker among apoptosis and cell survival in ccRCC patients. When this miR is downregulated it stimulates apoptosis, but if shows unchanged or higher levels compared with control it promotes the cell growth.
This hypothesis is also consistent with follow up data, therefore, the expression of miR501-5p could be considered as a new biomarker for the prognosis of clear cell renal carcinoma
Neurodegeneration induced by complexI inhibition in a cellular model of familial amyotrophic lateral sclerosis
No full textG93A Cu/Zn superoxide dismutase (SOD1), a human mutant SOD1 associated with familial amyotrophic lateral sclerosis, increased the toxicity of the mitochondrial toxin rotenone in the NSC-34 motoneuronal cell line. G93ASOD1 cells died more than untransfected and wild-type SOD1 cells after 6 and 24h exposure to 12.5 microM rotenone. Biparametric flow cytometry showed that rotenone induced rapid hyperpolarization of mitochondrial membrane potential (deltapsi(m)) in all the cell lines, followed by depolarization, and then by cell death. However, G93ASOD1 mitochondria were significantly more likely to shift from a hyperpolarized to a depolarized condition, and within the still viable cell population there was a higher proportion with depolarized mitochondria, a condition that can be envisaged as a commitment to cell death. ATP, which is needed to prevent loss of deltapsi(m), decreased more rapidly and to a greater extent in rotenone-treated G93ASOD1 cells than in the untransfected and wtSOD1cells. In all the cell lines, 1h after rotenone exposure, mitochondrial hyperpolarization was accompanied by the formation of a comparable amount of reactive oxygen species. However, G93ASOD1 cells reached the highest reactive oxygen species level since their basal level was higher than in untransfected and wild-type SOD1 cells. Our findings indicate that the mutant protein G93ASOD1 enhances the vulnerability of motor neurons to rotenone by mechanism(s) involving oxidative stress and perturbed mitochondrial homeostasis. This suggests that motor neurons from individuals carrying the mutant G93ASOD1 are at greater risk of death after inhibition of the electron transport chain
Berberine slows cell growth in autosomal dominant polycystic kidney disease cells
No full textAutosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary monogenic disorder characterized by development and enlargement of kidney cysts that lead to loss of renal function. It is caused by mutations in two genes (PKD1 and PKD2) encoding for polycystin-1 and polycystin-2 proteins which regulate different signals including cAMP, mTOR and EGFR pathways. Abnormal activation of these signals following PC1 or PC2 loss of function causes an increased cell proliferation which is a typical hallmark of this disease. Despite the promising findings obtained in animal models with targeted inhibitors able to reduce cystic cell growth, currently, no specific approved therapy for ADPKD is available. Therefore, the research of new more effective molecules could be crucial for the treatment of this severe pathology. In this regard, we have studied the effect of berberine, an isoquinoline quaternary alkaloid, on cell proliferation and apoptosis in human and mouse ADPKD cystic cell lines. Berberine treatment slows cell proliferation of ADPKD cystic cells in a dose-dependent manner and at high doses (100μg/mL) it induces cell death in cystic cells as well as in normal kidney tubule cells. However, at 10μg/mL, berberine reduces cell growth in ADPKD cystic cells only enhancing G0/G1 phase of cell cycle and inhibiting ERK and p70-S6 kinases. Our results indicate that berberine shows a selected antiproliferative activity in cellular models for ADPKD, suggesting that this molecule and similar natural compounds could open new opportunities for the therapy of ADPKD patients
Molecular basis for motor neuron selective damage induced by mutated SOD1: distribution of the G93A mutant of SOD1 in mitochondria and mitochondrial structural alterations in motor neuronal-like and in non-neuronal cellular models of ALS.
No full textMIR-501 DEPLETION INDUCES CELL CYCLE INHIBITION BY MTOR AND P53 MODULATION IN RENAL CARCINOMA
No full textINTRODUCTION: MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression at post-transcriptional level. The abnormal expression and mutation of miRs has been observed in most urologic cancers including renal cancer, thus they may contribute to development and progression of kidney carcinoma. In fact, their impaired function could trigger a series of altered signalling resulting in abnormal differentiation, proliferation and apoptosis. In the last years, is emerging the necessity to use miRs as biological biomarkers in order to improve diagnosis, prognosis and therapy response in renal carcinomas. Furthermore, miRNAs might be potential targets for novel therapeutic strategies, especially in patients with tumour subtypes that do not respond to currently available therapies (1-2).
Here, we have focalized our study on the role of miR501-5p in kidney carcinomas because it has been found differently expressed in kidney cancer tissues compared with normals of the same patients.
MATERIAL AND METHODS: Analysis of miR501-5p expression was performed by real time RT-PCR. Depletion or enrichment of this miR was conducted by specific antagomiRs and plasmid expressing miR-501-5p specific sequences, respectively. Protein activity was analyzed by immunological and cell imaging techniques. Apoptosis was studied through caspase-3 activity and cell cycle analysis was performed by propidium iodide staining.
RESULTS: We have analyzed the expression of miR501-5p in 36 clear cell (ccRCC) and 11 papillary (pRCC) kidney carcinomas. The expression of miR501-5p was higher in ccRCC (3.72 fold) and lower (3.76 fold) in pRCC tissues compared with normal kidneys derived from the same subjects, respectively. However, the distribution of miR501-5p expression values in ccRCC was found strongly variable. Follow up data of 25 ccRCC and 5 pRCC patients suggest that subjects with showed lower expression of miR-501 in cancer tissues respect to control (normal kidney), exhibited a good prognosis compared with patients with unchanged or high levels of this small RNA.
In order to evaluate the role of miR501-5p in renal cancer, we have depleted it by a specific antagomiR in KJ29 kidney cancer cell line (3). KJ29 cells expressed higher levels of miR501-5p than normal immortalized tubular kidney cells. The transfection of KJ29 cells with antagomiR caused a 50% reduction of miR501-5p expression compared with untransfected cells. Furthermore, the reduction of miR501-5p induced an increase in G0/G1 phase of cell cycle and a decrease of mTOR activity in KJ29 cells. In addition, the treatment with antagomiR caused an increase in caspase-3 activity, suggesting that this miR may regulate apoptosis. Moreover, miR-501-5p depletion enhanced the expression of p53, data also observed in kidney cancer tissues expressing lower levels of this miR than controls. The activation of p53 was also observed by its nuclear translocation in KJ29 treated with antagomiR. KJ29 cells were also transfected with a plasmid expressing miR-501-5p sequences and these cells showed an increased level of miR-501 compared with untranfected cells.
CONCLUSIONS: Our findings show that miR501-5p was differentially expressed in ccRCC. High or unchanged levels of miR501-5p seem not related with grading and metastasis in ccRCC, however, when it is downregulated could promote a good prognosis. Data reported suggest an anti apoptotic role for miR501-5p, making it a likely risk factor for a poor prognosis in renal carcinoma. Therefore, the expression of miR501-5p could be considered as potential biomarker for the prognosis of clear cell kidney carcinoma.
1. Schaefer A, Stephan C, Busch J, Yousef GM, Jung K: Diagnostic, prognostic and therapeutic implications of microRNAs in urologic tumors. Nat Rev Urol;7(5):286-97, 2010.
2. Cairns P: Renal cell carcinoma. Cancer Biomark; 9(1-6):461-73, 2010.
3. Del Senno et al. Cell Biology International Reports. 1986; 10:195
Detection of DNA mutations in urine of patients with prostate cancer by NGS technology
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