缺氧微环境SIRT1亚细胞定位对结直肠癌细胞凋亡的影响及其机制研究

目的:探究缺氧微环境SIRT1亚细胞定位对结直肠癌细胞凋亡的影响及其分子机制。方法:将编码过表达野生型SIRT1以及核定位序列(nuclear localization sequence,NLS)突变型SIRT1(SIRT1NLSmt)的慢病毒载体转染人类结肠癌HCT116细胞株,经嘌呤霉素筛选获得稳定过表达野生型SIRT1细胞株(LV-SIRT1细胞)和细胞质定位的NLS突变型SIRT1细胞株(LV-SIRT1NLSmt细胞),通过观察慢病毒载体编码的SIRT1-GFP融合蛋白的荧光定位,明确稳定转染细胞中外源性SIRT1的亚细胞定位。利用real-time PCR、Western blot法对分离提取的核-质蛋白进行检测,证实外源性SIRT1的表达和亚细胞定位情况。利用CCK-8细胞毒性实验、流式细胞术检测和TUNEL染色比较缺氧(1%O2)处理前后LV-SIRT1和LV-SIRT1NLSmt细胞存活或凋亡情况,Western blot法检测凋亡相关蛋白p53、ac-p53(K382)、Bcl-2、Bax、caspase-3和cleaved caspase-3表达水平。结果:Western blot、real-time PCR和免疫荧光染色结果显示稳定转染细胞均存在外源性SIRT1的过表达,NLS突变可导致SIRT1NLSmt富集于细胞质中;与亲本细胞HCT116和LV-SIRT1NLSmt细胞相比,LV-SIRT1细胞对缺氧的耐受能力最差、细胞凋亡水平最高,凋亡相关蛋白p53、Bax、caspase-3、cleaved caspase-3表达水平显著升高,ac-p53(K382)和Bcl-2表达水平显著下降,且LV-SIRT1细胞的胞核ac-p53下降最为显著。结论:在缺氧微环境中,细胞核定位的SIRT1通过影响p53的去乙酰化水平促进结直肠癌细胞凋亡。     

SIRT1 activation by resveratrol ameliorates cisplatin-induced renal injury through deacetylation of p53

Nephrotoxicity is one of the important dose-limiting factors during cisplatin treatment. There is a growing body of evidence that activation of p53 has a critical role in cisplatin-induced renal apoptotic injury. The nicotinamide adenine dinucleotide-dependent protein deacetylase SIRT1 decreases apoptosis through deacetylating of p53, and resveratrol is known as an activator of SIRT1. To study the role of SIRT1 in cisplatin-induced renal injury through interaction with p53, mouse proximal tubular cells (MPT) were treated with cisplatin and examined the expression level of SIRT1, acetylation of p53, PUMA-α, Bax, the cytosolic/mitochondrial cytochrome c ratio, and active caspase-3. The expression of SIRT1 was decreased by cisplatin. Resveratrol, a SIRT1 activator, ameliorated cisplatin-induced acetylation of p53, apoptosis, and cytotoxicity in MPT cells. In addition, resveratrol remarkably blocked cisplatin-induced decrease of Bcl-xL in MPT cells. Further specific SIRT1 inhibition with EX 527 or small interference RNA specific to SIRT1 reversed the effect of resveratrol on cisplatin-induced toxicity. Inhibition of p53 by pifithrin-α reversed the effect of EX527 in protein expression of PUMA-α, Bcl-xL, and caspase-3 and cytotoxicity in MPT cells. SIRT1 protein expression after cisplatin treatment was significantly decreased in the kidney. SIRT1 activation by resveratrol decreased cisplatin-induced apoptosis while improving the glomerular filtration rate. Taken together, our findings suggest that the modulation of p53 by SIRT1 could be a possible target to attenuate cisplatin-induced kidney injury.     

TGEV nucleocapsid protein induces cell cycle arrest and apoptosis through activation of p53 signaling

Our previous studies showed that TGEV infection could induce cell cycle arrest and apoptosis via activation of p53 signaling in cultured host cells. However, it is unclear which viral gene causes these effects. In this study, we investigated the effects of TGEV nucleocapsid (N) protein on PK-15 cells. We found that TGEV N protein suppressed cell proliferation by causing cell cycle arrest at the S and G2/M phases and apoptosis. Characterization of various cellular proteins that are involved in regulating cell cycle progression demonstrated that the expression of N gene resulted in an accumulation of p53 and p21, which suppressed cyclin B1, cdc2 and cdk2 expression. Moreover, the expression of TGEV N gene promoted translocation of Bax to mitochondria, which in turn caused the release of cytochrome c, followed by activation of caspase-3, resulting in cell apoptosis in the transfected PK-15 cells following cell cycle arrest. Further studies showed that p53 inhibitor attenuated TGEV N protein induced cell cycle arrest at S and G2/M phases and apoptosis through reversing the expression changes of cdc2, cdk2 and cyclin B1 and the translocation changes of Bax and cytochrome c induced by TGEV N protein. Taken together, these results demonstrated that TGEV N protein might play an important role in TGEV infection-induced p53 activation and cell cycle arrest at the S and G2/M phases and apoptosis occurrence.     

Hck Promotes Neuronal Apoptosis Following Intracerebral Hemorrhage

The hematopoietic cell kinase (Hck) is a member of the Src family protein kinases which regulates many signal transduction pathways including cell growth, proliferation, differentiation, migration, and apoptosis. However, the expression and function of Hck after intracerebral hemorrhage (ICH) are unknown. Western blot, immunohistochemistry, and immunofluorescence showed that Hck was obviously up-regulation in neurons adjacent to the hematoma after ICH. In addition, the temporary raise of Hck expression was paralleled with the expression of p53, Bax, and active caspase-3, suggesting that Hck was involved in neuronal apoptosis. Hck siRNA dramatically decrease hemin-induced expression of p53, Bax, and active caspase-3 as well as the amount of apoptotic SH-SY5Y cells in vitro. Furthermore, Hck interacted with p53. Hence, Hck might promote neuronal apoptosis via p53 signaling pathway after ICH.     

Functional proteomics of resveratrol-induced colon cancer cell apoptosis: caspase-6-mediated cleavage of lamin A is a major signaling loop

The study investigated the molecular basis of resveratrol (RSV)-evoked apoptosis in four (Bax+/-, Bax-/-, p53+/+, and p53-/-) HCT116 colon cancer cell lines. RSV induced apoptosis in all the cells in a dose-dependent manner; however, Bax+/- and p53+/+ cells were more susceptible than their knockout counterparts (Bax-/- and p53-/-, respectively). Using Bax+/- cells as a model, proteomic analysis revealed four RSV-responsive events: fragmentation of lamin A/C protein; increase in concentration of a more basic isoelectric variant of the ribosomal protein P0; and decrease in concentration of dUTPase as well as stathmin 1. Lamin A cleavage in response to RSV treatment was confirmed using Western blot analysis. Caspase-6 was activated, which was evidenced by cleavage and accumulation in active form of caspase-6 as well as upregulation of the protease activity. RSV-elicited lamin A cleavage and apoptosis were entirely abrogated by the peptide inhibitors of caspase-6. Likewise, partial knockdown of caspase-6 expression using small interfering RNA resulted in significant inhibition of RSV-elicited lamin A cleavage and apoptosis. Furthermore, the lower apoptosis sensitivity of the knockout cells (Bax-/- and p53-/-) correlated with the relatively reduced processing of caspase-6 and lamin A cleavage. Taken together, these data highlight the critical role of caspase-6 and its cleavage of lamin A in apoptotic signaling triggered by RSV in the colon carcinoma cells, which can be activated in the absence of Bax or p53.     

Apocynum venetum leaf extract protects rat cortical neurons from injury induced by oxygen and glucose deprivation in vitro

This study aimed to investigate the protective effect of Apocynum venetum leaf extract (AVLE) on an in vitro model of ischemia-reperfusion induced by oxygen and glucose deprivation (OGD) and further explored the possible mechanisms underlying protection. Cell injury was assessed by morphological examination using phase-contrast microscopy and quantified by measuring the amount of lactate dehydrogenase (LDH) leakage; cell viability was measured by XTT reduction. Neuronal apoptosis was determined by flow cytometry, and electron microscopy was used to study morphological changes of neurons. Caspase-3,?-8, and?-9 activation and Bcl-2/Bax protein expression were determined by Western blot analysis. We report that treatment with AVLE (5 and 50?μg/mL) effectively reduced neuronal cell death and relieved cell injury induced by OGD. Moreover, AVLE decreased the percentage of apoptotic neurons, relieved neuronal morphological damage, suppressed overexpression of active caspase-3 and?-8 and Bax, and inhibited the reduction of Bcl-2 expression. These findings indicate that AVLE protects against OGD-induced injury by inhibiting apoptosis in rat cortical neurons by down-regulating caspase-3 activation and modulating the Bcl-2/Bax ratio.     

MicroRNA-29a-3p regulates abdominal aortic aneurysm development and progression via direct interaction with PTEN

Various research studies have been conducted in deducing the role of microRNAs (miRNAs) in the pathogenesis and physiological processes of various systematic diseases. This study aims at demonstration of the important role played by miR-29a-3p, through association with phosphatase and tensin homolog (PTEN), in the regulation of abdominal aortic aneurysm development and progression. Quantitative real-time polymerase chain reaction (RT-qPCR) examined miRNA-19a-3p and PMEPA1 expression in multiplied vascular smooth muscle cells (VSMCs). Cell transfection upregulated or downregulated the genes and cell counting kit-8 assay determined cellular viability. RT-qPCR detected cellular proliferation and cell death using the cell proliferation and apoptosis biomarkers Ki87 and proliferating cell nuclear antigen, caspase-8 and caspase-3, respectively. Furthermore, luciferase assay analyzed the luciferase activity and western blot analysis determined miRNA-19a-3p and PMEPA1 protein expression in proliferation and apoptosis biomarkers. TargetScan 4.2 online software ( www.targetscan.org ) was used to perform the bioinformatics analysis so as to forecast the putative targets of miR-29a-3p and PTEN. The results inferred that there was an increased expression of miRNA-29a-3p found in AAA-mimic cells with increased cellular viability and significant pathological apoptosis. Further, when the expression of miRNA-29a-3p was downregulated, it reduced the cell viability of AAA cells. On the basis of the gene interplays, it can be understood that the PTEN was directly targeted by miRNA-29a-3p so as to regulate the AAA progression. Thus, PTEN was found to strengthen the proliferation effect of miRNA-29a-3p in AAA cells. The current study thus shed more insights about the molecular mechanistic roles of miRNA-29a-3p and PTEN, opening doors for novel therapeutic approach to AAA.     

Oridonin-induced mitochondria-dependent apoptosis in esophageal cancer cells by inhibiting PI3K/AKT/mTOR and Ras/Raf pathways

Oridonin, an active diterpenoid isolated from Rabdosia rubescens, has been reported for its antitumor activity on several cancers. However, its effect on human esophageal cancer remains unclear. In this study, we demonstrated that oridonin could inhibit the growth of human esophageal cancer cells both in vitro and in vivo. Oridonin not only suppressed the proliferation, but also induced cell cycle arrest and mitochondrial-mediated apoptosis in KYSE-30, KYSE-150, and EC9706 cells with dose-dependent manner. Further mechanism studies revealed that oridonin led cell cycle arrest in esophageal cancer cells via downregulating cell cycle-related proteins, such as cyclin B1 and CDK2, while upregulating p53 and p21. Oridonin also increased proapoptotic protein Bax and reduced antiapoptotic protein Bcl-2, as well as the increased expression of cleaved caspase-3, -8, and -9. In addition, oridonin treatment could significantly inhibit the PI3K/Akt/mTOR and Ras/Raf signaling pathway. In vivo results further demonstrated that oridonin treatment markedly inhibited tumor growth in the esophageal cancer xenograft mice model. Taken together, these results suggest that oridonin may be a potential anticancer agent for the treatment of esophageal cancer.     

HSP25 down-regulation enhanced p53 acetylation by dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis

Heat shock proteins (HSPs) play important roles in cellular stress resistance. Previous reports had already suggested that HSP27 played multiple roles in preventing doxorubicin-induced cardiotoxicity. Although HSP25 might have biological functions similar to its human homolog HSP27, the mechanism of HSP25 is still unclear in doxorubicin-induced cardiomyocyte apoptosis. To investigate HSP25 biological function on doxorubicin-induced apoptosis, flow cytometry was employed to analyze cell apoptosis in over-expressing HSP25 H9c2 cells in presence of doxorubicin. Unexpectedly, the H9c2 cells of over-expressing HSP25 have no protective effect on doxorubicin-induced apoptosis. Moreover, no detectable interactions were detected by coimmunoprecipitation between HSP25 and cytochrome c, and HSP25 over-expression failed in preventing cytochrome c release induced by doxorubicin. However, down-regulation of endogenous HSP25 by a specific small hairpin RNA aggravates apoptosis in H9c2 cells. Subsequent studies found that HSP25, but not HSP90, HSP70, and HSP20, interacted with SIRT1. Knockdown of HSP25 decreased the interaction between SIRT1 and p53, leading to increased p53 acetylation on K379, up-regulated pro-apoptotic Bax protein expression, induced cytochrome c release, and triggered caspase-3 and caspase-9 activation. These findings indicated a novel mechanism by which HSP25 regulated p53 acetylation through dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis.     

Mir-335-5p靶向G6PD对结肠癌细胞增殖、凋亡的影响

目的:探讨Mir-335-5p通过靶向G6PD对结肠癌细胞增殖、凋亡的影响.方法:设置正常结肠细胞组、空白对照组、NC组、miRNA-335-5p mimic组;体外培养结肠上皮细胞(IEC)和人源性结肠癌细胞SW480,并对NC组、miRNA-335-5p mimic组细胞进行转染;采用RT-qPCR检测各组细胞中m...     

SIRT1 is upregulated in cutaneous T-cell lymphoma,and its inhibition induces growth arrest and apoptosis

Silent information regulator type-1 (SIRT1) is the best-studied member of the Sirtuin (Sir2) family of nicotinamide dinucleotide (NAD)-dependent class III histone deacetylases (HDACs), but has not yet been explored in cutaneous T-cell lymphoma (CTCL). We analyzed five CTCL cell lines and lesional tissues using flow cytometry, immunostaining, immunoblotting, cell death, viability, and apoptosis assays, small-molecule inhibitors, and shRNA knockdown. We found strong SIRT1 expression among CTCL lines relative to normal lymphocytes. CTCL cells in lesional tissues also expressed SIRT1 strongly. SIRT1 knockdown resulted in reduced cellular metabolism and proliferation, increased apoptosis, and PARP cleavage products. Tenovin-1, which reversibly inhibits class III HDACs (SIRT1 and SIRT2), reduced SIRT enzymatic activity and SIRT1 expression and led to increased apoptosis. These alterations were accompanied by increased forkhead box O3 (FoxO3) in several cell lines and increased nuclear p53, as well as acetylated p53 in wtp53 MyLa CTCL line. A combination of class I/II and class III HDACIs (vorinostat and tenovin-1) produced significantly greater growth inhibition, cell death via apoptosis, as well as superior p53 promoter upregulation in wtp53 MyLa cells as compared with either agent alone. This occurred in a partially p53-dependent manner, as these effects were blunted by p53 knockdown. Our results indicate that SIRT1 is strongly expressed in CTCL. Its inhibition results in reduced growth and increased apoptosis of CTCL cells. Furthermore, our findings suggest that some CTCL patients, such as those with wtp53, might benefit more from treatment with a combination of different classes of HDACIs than with a single agent.     

Geldanamycin analog 17-DMAG limits apoptosis in human peripheral blood cells by inhibition of p53 activation and its interaction with heat-shock protein 90 kDa after exposure to ionizing radiation

Exposure to ionizing radiation induces p53, and its inhibition improves mouse survival. We tested the effect of 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG) on p53 expression and function after radiation exposure. 17-DMAG, a heat-shock protein 90 (Hsp90) inhibitor, protects human T cells from ionizing radiation-induced apoptosis by inhibiting inducible nitric oxide synthase (iNOS) and subsequent caspase-3 activation. Using ex vivo human peripheral blood mononuclear cells, we found that ionizing radiation increased p53 accumulation, acute p53 phosphorylation, Bax expression and caspase-3/7 activation in a radiation dose- and time postirradiation-dependent manner. 17-DMAG inhibited these increases in a concentration-dependent manner (IC(50) = 0.93 ± 0.01?μM). Using in vitro models, we determined that inhibition of p53 by genetic knockout resulted in lower levels of caspase-3/7 activity 1?day after irradiation and enhanced survival at 10?days. Analysis of p53-Hsp90 interaction in ex vivo cell lysates indicated that the binding between the two molecules occurred after irradiation but 17-DMAG prevented the binding. Taken together, these results suggest the presence of p53 phosphorylation and Hsp90-dependent p53 stabilization after acute irradiation. Hsp90 inhibitors such as 17-DMAG may prove useful with radiation-based cancer therapy as well as for general radioprotection.     

MicroRNA-543 promotes ovariectomy-induced osteoporosis through inhibition of AKT/p38 MAPK signaling pathway by targeting YAF2

The present study aimed to determine the roles of miRNA-543 in osteoporosis in rats induced by ovariectomy. The osteoporosis rat model was established by ovariectomy induction. MiRNA-543 expression in osteoblasts was measured by quantitative real-time polymerase chain reaction. The cell proliferation and apoptosis were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry assays, respectively. Western blot analysis was conducted to examine the expression of YAF-2 and AKT signaling. TargetScan analysis and dual-luciferase reporter assay were performed to determine the target gene of miRNA-543. MiRNA-543 was significantly upregulated in osteoporosis rat model. Overexpression of miRNA-543 significantly suppressed cell growth and promoted apoptosis in osteoblasts, whereas downregulation of miRNA-543 significantly enhanced cell growth and inhibited apoptosis. MiRNA-543 upregulation significantly inhibited YAF-2 expression and suppressed the phosphorylation and expression of AKT and p38 mitogen-activated protein kinases (MAPK) in osteoblasts. Furthermore, YAF-2 knockdown enhanced the effects of miRNA-543 on apoptosis in osteoblasts. AKT inhibitor MK2206 and p38 MAPK inhibitor SB203580 also enhanced the effects of miRNA-543 on apoptosis in osteoblasts. Our findings revealed that inhibition of miRNA-543 could protect osteoblasts against ovariectomy-induced osteoporosis through AKT/p38 MAPK signaling pathway by targeting YAF2.     

瞬时受体电位香草酸亚型1激活释放的P物质在小鼠急性心肌梗死后凋亡中的保护作用

瞬时受体电位香草酸亚型1 (transient receptor potential vanilloid 1, TRPV1)在心肌缺血激活后可传导心绞痛信号和释放P物质(substance P, SP).SP是速激肽家族成员之一,主要通过结合并激活神经激肽1 (neurokinin 1,NK1)受体发挥作用. TRPV1和SP在缺血性心脏病中对心功能的恢复和重塑有一定保护作用,但对心肌梗死后凋亡的作用及具体机制尚不明确.本研究用TRPV1基因敲除(TRPV1-/- )小鼠和野生型(wide type, WT)小鼠建立心肌梗死模型,并外源性给予SP和NK1受体拮抗剂RP67580,用TTC染色法观察梗死的面积,TUNEL法检测心肌细胞凋亡指数,Western印迹方法检测caspase-3、Bcl-2、Bax、p53的蛋白表达.结果发现,心肌梗死24 h后,TRPV1-/-小鼠比WT小鼠梗死面积更大,凋亡指数和caspase-3活性更高,Bcl-2/Bax和p53蛋白表达更低. SP预处理可以明显缩小TRPV1-/-小鼠梗死面积,降低凋亡指数、caspase-3活性和升高Bcl-2/Bax比值,而在WT小鼠中改善不明显.外源性给予RP67580,阻断SP与NK1受体结合后,与相应对照组相比,WT小鼠梗死面积和凋亡指数更大,caspase-3蛋白表达更高,Bcl-2/Bax比值更低;TRPV1-/-小鼠与相应对照组比较,凋亡指数和caspase-3表达升高,Bcl-2/Bax比值降低.研究结果表明,SP可能介导了TRPV1在急性心肌梗死后凋亡中的保护作用.