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Pathological cardiac hypertrophy serves as a significant foundation for cardiac dysfunction and heart failure. Recently, growing evidence has revealed that microRNAs (miRNAs) play multiple roles in biological processes and participate in cardiovascular diseases. In the present research, we investigate the impact of miRNA-34c-5p on cardiac hypertrophy and the mechanism involved. The expression of miR-34c-5p was proved to be elevated in heart tissues from isoprenaline (ISO)-infused mice. ISO also promoted miR-34c-5p level in primary cultures of neonatal rat cardiomyocytes (NRCMs). Transfection with miR-34c-5p mimic enhanced cell surface area and expression levels of foetal-type genes atrial natriuretic factor (Anf) and β-myosin heavy chain (β-Mhc) in NRCMs. In contrast, treatment with miR-34c-5p inhibitor attenuated ISO-induced hypertrophic responses. Enforced expression of miR-34c-5p by tail intravenous injection of its agomir led to cardiac dysfunction and hypertrophy in mice, whereas inhibiting miR-34c-5p by specific antagomir could protect the animals against ISO-triggered hypertrophic abnormalities. Mechanistically, miR-34c-5p suppressed autophagic flux in cardiomyocytes, which contributed to the development of hypertrophy. Furthermore, the autophagy-related gene 4B (ATG4B) was identified as a direct target of miR-34c-5p, and miR-34c-5p was certified to interact with 3' untranslated region of Atg4b mRNA by dual-luciferase reporter assay. miR-34c-5p reduced the expression of ATG4B, thereby resulting in decreased autophagy activity and induction of hypertrophy. Inhibition of miR-34c-5p abolished the detrimental effects of ISO by restoring ATG4B and increasing autophagy. In conclusion, our findings illuminate that miR-34c-5p participates in ISO-induced cardiac hypertrophy, at least partly through suppressing ATG4B and autophagy. It suggests that regulation of miR-34c-5p may offer a new way for handling hypertrophy-related cardiac dysfunction.
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Parkinson's disease (PD), known as one of the most universal neurodegenerative diseases, is a serious threat to the health of the elderly. The current treatment has been demonstrated to relieve symptoms, and the discovery of new small-molecule compounds has been regarded as a promising strategy. Of note, the homeostasis of the autolysosome pathway (ALP) is closely associated with PD, and impaired autophagy may cause the death of neurons and thereby accelerating the progress of PD. Thus, pharmacological targeting autophagy with small-molecule compounds has been drawn a rising attention so far. In this review, we focus on summarizing several autophagy-associated targets, such as AMPK, mTORC1, ULK1, IMPase, LRRK2, beclin-1, TFEB, GCase, ERR
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The hepatic endoplasmic reticulum (ER)-anchored cytochromes P450 (P450s) are mixed-function oxidases engaged in the biotransformation of physiologically relevant endobiotics as well as of myriad xenobiotics of therapeutic and environmental relevance. P450 ER-content and hence function is regulated by their coordinated hemoprotein syntheses and proteolytic turnover. Such P450 proteolytic turnover occurs through a process known as ER-associated degradation (ERAD) that involves ubiquitin-dependent proteasomal degradation (UPD) and/or autophagic-lysosomal degradation (ALD). Herein, on the basis of available literature reports and our own recent findings of as well as experimental studies, we discuss the therapeutic and pathophysiological implications of altered P450 ERAD and its plausible clinical relevance. We specifically (i) describe the P450 ERAD-machinery and how it may be repurposed for the generation of antigenic P450 peptides involved in P450 autoantibody pathogenesis in drug-induced acute hypersensitivity reactions and liver injury, or viral hepatitis; (ii) discuss the relevance of accelerated or disrupted P450-ERAD to the pharmacological and/or toxicological effects of clinically relevant P450 drug substrates; and (iii) detail the pathophysiological consequences of disrupted P450 ERAD, contributing to non-alcoholic fatty liver disease (NAFLD)/non-alcoholic steatohepatitis (NASH) under certain synergistic cellular conditions.
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Polo-like kinase (PLK1) has been identified as a potential target for cancer treatment. Although a number of small molecules have been investigated as PLK1 inhibitors, many of which showed limited selectivity. PLK1 harbors a regulatory domain, the Polo box domain (PBD), which has a key regulatory function for kinase activity and substrate recognition. We report on 3-bromomethyl-benzofuran-2-carboxylic acid ethyl ester (designated: MCC1019) as selective PLK1 inhibitor targeting PLK1 PBD. Cytotoxicity and fluorescence polarization-based screening were applied to a library of 1162 drug-like compounds to identify potential inhibitors of PLK1 PBD. The activity of compound MC1019 against the PLK1 PBD was confirmed using fluorescence polarization and microscale thermophoresis. This compound exerted specificity towards PLK1 over PLK2 and PLK3. MCC1019 showed cytotoxic activity in a panel of different cancer cell lines. Mechanistic investigations in A549 lung adenocarcinoma cells revealed that MCC1019 induced cell growth inhibition through inactivation of AKT signaling pathway, it also induced prolonged mitotic arrest-a phenomenon known as mitotic catastrophe, which is followed by immediate cell death apoptosis and necroptosis. MCC1019 significantly inhibited tumor growth in a murine lung cancer model without affecting body weight or vital organ size, and reduced the growth of metastatic lesions in the lung. We propose MCC1019 as promising anti-cancer drug candidate.
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Natural product evodiamine and its derivatives represent a promising class of multi-target antitumor agents. However, the clinical development of these compounds has been hampered by a poor understanding of their antitumor mechanisms. To tackle this obstacle, herein, novel fluorescent probes were designed to elucidate the antitumor mode of action of 10-hydroxyevodiamine. This compound was proven to be distributed in the mitochondria and lysosomes and to act by autophagy and apoptosis mechanisms.
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Objective: To investigate the effect of 3-methyladenine (3-MA) on autophagy, migration and invasion of epithelial ovarian cancer (EOC) cells under hypoxia. Methods: Different concentrations of 3-MA were used to treat EOC cells in hypoxic environment. The expression of autophagy-associated protein LC3 was detected by Western blotting. The autophagosome formation was observed by transmission electron microscopy. The proliferation, adhesion, migration and invasion of EOC cells were determined by thiazole blue colorimetry, adhesion test, Transwell migration test and Matrigel invasion test, respectively. Results: 3-MA was able to inhibit the increase of LC3- Ⅱ and autophagosome formation induced by hypoxia. In hypoxic environment, the survival rate of EOC cells was significantly decreased by 5.00 mmol/L 3-MA for 24 h (P=0.000). The adhesion ability of EOC cells was significantly decreased by 2.50 mmol/L 3-MA for 6 h (P=0.007). 1.25 mmol/L and 2.50 mmol/L 3-MA could inhibit the migration and invasion of EOC cells in hypoxic environment (P<0.01). Conclusion: 3-MA can inhibit the autophagy, migration and invasion of EOC cells in hypoxic environment.
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Objective To explore whether exendin-4 inhibits AR42J cells and its mechanism.Methods AR42J cells were treated with exendin-4 under multiple concentrations(1,5,10 pmol/L) at 24,48,72,96,120 h to assess its cell viability by MTT assay and got the IC-50 and time points.Then checking whether exendin-4 could induce the AR42Jcells apoptosis by setting normal control (NC) group,exendin-4 (Ex-4) group and Ex-4+ z-VAD-fmk (apoptosis inhibitor) group,and exploring whether 3-MA which is autophagy inhibitor could inhibit the AR42J cells apoptosis induced by exendin-4 by setting NC group,Ex-4 group and Ex-4+3-MA group.Cell viability was analyzed by MTT and the cells apoptosis was detected by flow cytometry and the protein levels of caspase-3,LC3 and p62 were studied by Western blot.Results Concentration of 10 pmol/L exendin-4 and and time point 72 h were selected for the further study.z-VAD-fmk pretreatment can significantly inhibit the cell viability of exendin-4 by (81.2±3.3)% vs.(49.4±3.0)% (P<0.05).Flow cytometry showed that exendin-4 could induce the AR42J cells apoptosis by (28.2± 1.4)% vs.(3.6±0.8)%,and increased the caspase-3 level by Western blot,which both can be reversed by (79.1 ±2.3) % vs.(49.8±2.5)% (P<0.05) when the cells were treated for 72 h,as was apoptosis ratio by (14.5±2.1)% vs.(29.2±3.2)%.Western blot showed that exendin-4 can upregulate protein levels of LC3B-Ⅱ,p62 和 caspase-3 and 3-MA,and pretreatment can inhibit the upregulation of LC3B-Ⅱ and caspase-3 but further increased the upregulation of p62 induced by exendin-4.Conclusions Exendin-4 can induce AR42J cells apoptosis and 3-MA pretreating can inhibit exendin-4 cytotoxicity through downregulating autophagy.So auto phagy inhibitor 3-MA could potentially extenuate the cytotoxicity of exendin-4 in pancreatic acinar cells.
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Objective To investigate the effects of rapamycin which is an autophagy inducer and 3-adenine (3-MA) which is an autophagy inhibitor on motor behavior and autophagy related protein LC3 in C57BL/6 mice of Parkinson's disease(PD).Methods 40 C57BL/6 male mice were randomly divided into control group and experimental groups which consist of MPTP model group,rapamycin group and 3-MA group,with 10 in each group.Mice in experimental groups received intraperitoneal injection with MPTP to establish PD models,and mice in control group received intraperitoneal injection with the same amount of saline solution for 1 week.Mice in rapamycin group received intraperitoneal injection with rapamycin and mice in 3-MA group received intraperitoneal injection with 3-MA at the same time when MPTP was injected.Animal behavior detections were carried out on the 1 th,7th and 14th day after the last injection.After the last injection,mice were sacrificed and sections of midbrain nigra were gained for the detection of expression of autophagy specificity protein LC3 by Western Blot.Results Compared with MPTP model group,rapamycin could improve the general condition and behavioral manifestation of mice in pole test((14.89± 1.14) s vs (16.24±1.39) s,P<0.05;(15.18±1.36) s vs(17.93±1.11s),P<0.01),traction test((1.7±0.5) vs (1.2±0.4),P< 0.05;(1.5±0.5)vs (1.1±0.3),P<0.05) and open field test which contained total activity distance((5 875.3 ± 1148.9) cm vs (5 061.5±773.1) cm,P<0.05;(5 753.2± 1 106.7) cm vs (4 669.3±969.1) cm,P<0.01) and average speed((19.29±2.35) cm/s vs (16.47±3.01) cm/s,P<0.05;(18.71±2.71)cm/s vs (15.80±2.50) cm/s,P<0.01),while 3-MA aggravated behavioral deficits of mice in pole test(19.92± 1.61s vs 17.93± 1.11 s,P<0.05),and both total activity distance((3 879.7±575.0) cm vs (4 669.3±969.1) cm,P<0.05) and average speed((13.34± 1.87) cm/s vs (15.80±2.50) cm/s,P<0.05) in open field test were decreased.The results of Western Blot confirmed that rapamycin could increase the expression of LC3-Ⅱ,however 3-MA could re duce the expression of LC3-Ⅱ.Conclusion This study confirmed that rapamycin could alleviate behavioral symptoms of PD model mice and increase the level of LC3 in midbrain nigra,while 3-MA could exacerbate behavioral symptoms in PD model mice and decrease the level of LC3 in midbrain nigra.Thus it can be speculated that drugs which can regulate autophagy may be potential treatment protocols for PD.
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Objective To explore effect of isoorientin on gastric cancer cell autophagy.Methods Isoorientin and autophagy activator and effect of inhibitors on proliferation of human gastric cancer cell line SGC-7901 by MTT assay.Cell apoptosis was detected by flow cytometry.Production of autophagy lysosomal in gastric cancer SGC-7901 cells was obseroved by AO and MDC staining.Characteristic expression of autophagy protein was analysed by Western blot.Results MTT assay indicated that isoorientin could inhibit gastric cancer cell growth, RAP has the same effects, but 3-MA inhibition of apoptosis.Flow cytometry was used to detect the apoptosis rate of isoorientin and RAP could promote cell apoptosis , while 3-MA had the opposite effect.In AO staining, the red light appeared in the cells, and the green fluorescence appeared in the cells of MDC staining, which showed that there was an autophagy in the cells.Western blot test found the isoorientin was cell autophagy specific protein LC-3II, Beclin-1 expression increased, 3-MA suppressed the expression of the two proteins, and RAP had the opposite effect.Conclusion Isoorientin could induce apoptosis in gastric cancer SGC-7901 cells, autophagy is involved in the process of death.
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Quercetin is a natural flavonoid phytochemical that is extracted from various plants. Having an advantages due to its varied biological properties, such as anti-inflammatory, anti-viral, anti-oxidant, and anti-cancer effects, quercetin is used to treat many diseases. Recently, it has been reported that autophagy inhibition may play a key role in anti-cancer therapy. Therefore, in this study, we investigated the molecular mechanisms and anti-cancer effects of quercetin in human osteosarcoma cells via autophagy inhibition. We ascertained that quercetin inhibited cell proliferation and induced cell death, these process is demonstrated that apoptosis via the mitochondrial pathway and the caspase cascade. Quercetin also induced autophagy which was inhibited by 3-MA, autophagy inhibitor and the blockade of autophagy promoted the quercetin-induced apoptosis, confirming that autophagy is a pro-survival process. Thus, these findings demonstrate that quercetin is an effective anti-cancer agent, and the combination of quercetin and an autophagy inhibitor should enhance the effect of anti-cancer therapy.
الموضوعات
Humans , Apoptosis , Autophagy , Cell Death , Cell Proliferation , Osteosarcoma , Quercetinالملخص
[Objective] To observe the effects of autophagy induced by different doses of ultraviolet B (UVB) irradiation on the apeptosis in human skin fibroblasts.[Methods] Skin fibroblasts were isolated from the circumcision specimen of a 23-year-old healthy male,and subjected to a primary culture.After 3 to 10 passages,the cells were collected and applied in the following experiment.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of some fibroblasts treated with different concentrations (0,0.5,2.0,5.0and 10.0 mmol/L) of 3-methyladenine (3-MA).To qualitatively and quantitatively detect the autophagy in fibroblasts treated with different concentrations of 3-MA and in fibroblasts treated with 3-MA of 0.5 mmol/Lfollowing UVB irradiation,monodansylcadaverine (MDC) staining was carried out,and immunofluorescence was used to detect the expression of microtubule-associated protein 1 light chain (LC3).Some fibroblasts were classified into 8 groups to remain untreated,be irradiated with UVB of 30,50 and 100 mJ/cm2 alone,treated with 3-MA of 0.5 mmol/L alone,or treated with 0.5 mmol/L 3-MA following irradiation with UVB of 30,50 and 100 mJ/cm2,respectively,then,cell apoptosis was qualitatively detected by Hoechst and propidium iodide (PI)staining,and quantitatively detected by flow cytometry with annexin V and PI.[Results] The percentage of autophagic cells was (63.037 ± 5.876) % in fibroblasts treated with starvation condition,significantly decreased to (34.425 ± 5.183) % in fibroblasts treated with 3-MA of 0.5 mmol/L.The expression of LC3 showed a gradually increasing trend from untreated fibroblasts,to fibroblasts irradiated with UVB of 30,50 and 100 mJ/cm2,while the increase was attenuated by the 4-hour treatment with 3-MA immediately after the irradiation.Compared with the other concentrations,the 3-MA of 0.5 mmol/L showed the least influence on the viability of fibroblasts.The addition of 3-MA of 0.5 mmol/L increased the percentage of cells both positive for Hoechst and PI staining in fibroblasts irradiated with UVB of 50 mJ/cm2,but decreased that in fibroblasts irradiated with UVB of 100 mJ/cm2.Similarly,the percentage of middle and late apoptotic cells was significantly higher in fibroblasts irradiated with UVB of 50 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 50 mJ/cm2alone ((10.933 ± 0.839) % vs.(7.267 ± 0.473) %,t =5.20,P< 0.05),but lower in fibroblasts irradiated with UVB of 100 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 100 mJ/cm2alone ( (7.100 ± 0.781 ) % vs.( 1 0.133 ± 0.681 ) %,t =6.29,P < 0.05 ).[Conclusion]s The irradiation with UVB of 50 mJ/cm2 may protect fibroblasts by inducing autophagy and suppressing apoptosis,while the high level of autophagy induced by UVB of 100 mJ/cm2 may lead to autophagic cell death in fibroblasts.
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ObjectiveTo explore the intervention effect and the underlying molecular mechanism of 3-Methyladenine on behavioral damage of neonatal rat with prolonged seizures. MethodsForty-five 6-dayold SD rats were randomly ( random number) divided into the recurrent prolonged neonatal seizure group ( RS group), the 3-MA-treated seizure group and control group. The volatile agent flurothyl was used to induce 30 min seizure attack. At postnatal day 6 (P6), recurrent seizures were induced once daily for successive 6 days in both RS group and 3-MA group. In 3-MA group, 3-MA (2 μL) was injected daily before seizures induced.Neural-behavior changes were observed with double-blind method including swimming development, open field test and Morris water maze analysis. Bcl-2 and Beclinl protein levels in hippocampus were detected by western blot method at P50. ResultsThe total scores of swimming behavior in RS rats were decreased significantly compared with those of control and 3-MA rats ( control: 7. 44 ±1. 13, RS: 5.06±1.63, 3-MA: 7.33 ±1.08, F=16.19, P<0. 01) . The start-latency time of open filed behavior in RS rats ( 13. 33 ±6. 69) were increased significantly compared with that of control (7. 11 ±2. 37) and 3-MA rats (9. 91 ±4. 23) (F=4. 39, P<0. 05). The escape latency was significantly longer in rats of RS group than that of control and 3-MA rats on the 4th and 5th days (P < 0.05). The level of Bcl-2 in hippocampus of RS group (0. 587 +0. 139) were significantly lower than that of control (0. 782 +0. 083) and 3-MA groups (0. 799 + 0. 163) (F =4. 7 1, P < 0. 05 ). There were no significant differences in the level of Beclin1 protein in hippocampus among the three groups ( F =0. 27, P > 0. 05 ).Conclusions Pretreatment with autophagy inhibitor 3-MA in acute phase of neonatal seizures could significantly improve neurobehavioral capacity, which might be associated with the increased in the level of Bcl-2 protein in hippocampus.
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Objective To investigate the effects of sirolimus and 3-methyladenine (3-MA) on the autophagy in,as well as matrix matalloproteinase (MMP)-1 and MMP-3 secretion by,human skin fibroblasts (HSFs).Methods HSFs were isolated from the circumcised foreskin of a healthy male,and subjected to primary culture.After 3 to 10 passages,HSFs were incubated with sirolimus of 20,50,100,250 nmol/L and 3-MA of 0.5,2.0,5.0,10.0 mmol/L respectively for 4 hours followed by the observation of autophagy and detection of MMP-1 and MMP-3 levels in the supernatant by enzyme linked immunosorbent assay (ELISA).Those HSFs remaining untreated or treated with dimethyl sulfoxide served as the control.Results The percentage of autophagy-positive cells was 59.075% ± 6.884%,76.350% ± 5.226%,85.063% ± 6.002%,86.288% ± 5.558% and 96.825% ± 1.500% respectively in HSFs treated with sirolimus of 0,20,50,100 and 250 nmol/L; significant differences were observed between the 5 groups (P < 0.01 ) but not between the cells treated with sirolimus of 50 and 100 nmol/L (P > 0.05).After being treated with 3-MA of 0,0.5,2.0,5.0 and 10.0 mmol/L,the percentage of autophagy-positive cells in HSFs was 63.037% ± 5.876%,34.425% ± 5.183%,19.700% ± 3.028%,12.900% ± 3.334% and 7.775% ± 2.293% respectively with a significant difference between these groups (all P < 0.01 ).Elevated levels of MMP-1 and MMP-3 were observed in the supernatant of HSFs treated with sirolimus of 250 nmol/L and 3-MA of 10.0 mmol/L (all P < 0.05).Conclusions The autophagy in HSFs can be upregulated by sirolimus,but downregulated by 3-MA.For the secretion of MMPs by HSFs,3-MA and high concentrations of sirolimus exert a promotive effect,and the effect of 3-MA is in a concentration-related manner,but the influences of sirolimus at lower concentrations remain unclear.
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Researches have shown that melatonin is neuroprotectant in ischemia/reperfusion-mediated injury.Although melatonin is known as an effective antioxidant,the mechanism of the protection cannot be explained merely by antioxidation.This study was devoted to explore other existing mechanisms by investigating whether melatonin protects ischemia/reperfusion-injured neurons through elevating autophagy,since autophagy has been frequently suggested to play a crucial role in neuron survival.To find it out,an ischemia/reperfusion model in N2a cells was established for examinations.The results showed that autophagy was significantly enhanced in N2a cells treated with melatonin at reper-fusion onset following ischemia and greatly promoted cell survival,while autophagy blockage by 3-MA led to the shortened N2a cell survival as assessed by MTT,transmission electron microscopy,and laser confocal scanning microscopy.Besides,the protein levels of LC311 and Beclinl were remarkably increased in ischemia/reperfusion-injured N2a in the presence of melatonin,whereas the expression of p-PKB,key kinase in PI3K/PKB signaling pathway,showed a decrease when compared with untreated subjects as accessed by immunoblotting.Taken together these data suggest that autophagy is possibly one of the mechanisms underlying neuroprotection of melatonin.
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Objective To explore the expressions of autophagy-related gene Beclin-1 and LC3 in adriamycin induced eardiomyopathy rats,to prnve that autophagy might take part in the development of adriamycin induced eardiomyopathy in rats,so as to provide experimental and theoretical evidence for preventing and treating adriamycin induced cardiomyopathy.Methods Forty-five male SD rats were randomly divided into 3 groups,control groups,ADR group and ADR+3-MA group.The model of adriamycin induced cardiomyopathy rats was established.The tissue sample taken from the left ventricle wall was checked for the morphons of autophagosome by electron microscope. The expressions of Beelin-1 and LC3 of myocardium were detected.Results The morphons of autophagosome in ADR group was significantly increased compared with that in control and ADR +3-MA groups. The expression of Beclin-1 in myocardium of ADR group was significantly inereased compared with that in control and ADR +3-MA groups (P < 0.05).The level of LC3 in myocardium of ADR group was significantly increased compared with that in control and ADR+3-MA groups (P < 0.05).Conclusions Autophagy plays an important role in adriamycin induced cardiomyopathy.
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Objective To explore the repetitive expressions of autophagy marker protein-rnicrotubule-associ-ated protein 1 light chain 3 (LC3) in hippocampus in newborn rats with recurrent seizure and the influence of 3-methyladeine (3-MA) on LC2 expressions. Method Seventy-two 6-day-old SD rats were randomly (random nam-ber) divided into the recurrent neonatal seizure group (RS group, n = 24), the 3-MA-treated seizure group (3-MA group, n = 24) and control group (n = 24). Rats in RS group were subjected to 55 attacks of seizure induced by flurothyl in 9 successive days from the 6th postnatal day (P6). In 3-MA group, 2 μL of 3-MA was injected every day till seizure induced. Western blot analysis was used to determine LC3 protein level in hippocampus at different intervals of 1.5 h,3 h,6 h and 24 h after the last convulsion. The LC3 protein level was analyzed with Dunnett test after ANOVA. Results LC3 protein levels in RS group at the different intervals were significantly higher than those in the control group and in 3-MA group (F =4.70,5.28,8.51 and 5.89, respectively, P <0.05), and there were no significant differences in LC3 protein level between 3-MA group and control group at those intervals (P > 0.05). Conclusions The autophagy/lysosomal pathway is immediately activated after recurrent seizure evidenced by the elevated expressions of LC3 in hippocampus. The 3-MA is involved in the regulation of autophagy/ lysosomal pathway by down-regulating the expressions of LC3.