Multiple actions of pifithrin-alpha on doxorubicin-induced apoptosis in rat myoblastic H9c2 cells

Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-alpha (PFT-alpha), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 muM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-alpha followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-alpha also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-alpha interferes with multiple steps of Dox-induced apoptosis.     

Angiostatin antagonizes the action of VEGF-A in human endothelial cells via two distinct pathways

Angiostatin consisting of the first four-kringle domains of the plasminogen potently inhibits angiogenesis in vitro and in vivo. However, the molecular mechanism of action whereby angiostatin mediates its inhibitory effect on proliferating endothelial cells remains elusive. We therefore used the proliferating cultured human umbilical vein endothelial cells (HUVECs) promoted by vascular endothelial growth factor A to identify the endogenous signaling elements that mediate the antiangiogenic effect of angiostatin. Treatment of HUVEC with angiostatin at a concentration known to inhibit cell proliferation and induce apoptosis resulted in induction of p53-, Bax-, and tBid-mediated release of cytochrome c into the cytosol. In addition, angiostatin also activated the Fas-mediated apoptotic pathway in part via up-regulation of FasL mRNA, down-regulation of c-Flip, and activation of caspase 3. These results suggest that the anti-angiogenic action of angiostatin is likely mediated by two distinct signaling pathways, one intrinsic mediated by p53 while the other extrinsic involved in FasL engagement and mitochondria dysfunction.     

Different mechanisms of saturated versus polyunsaturated FFA-induced apoptosis in human endothelial cells

Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 micromol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.     

Pifithrin-alpha, an inhibitor of p53, enhances the genetic instability induced by etoposide (VP16) in human lymphoblastoid cells treated in vitro

Recent studies indicate that p53-dependent apoptosis induced in normal tissues during chemo- and radiotherapy can cause severe side effects of anti-cancer treatments that limit their efficiency.The aim of the present work was to further characterise the role of p53 in maintaining genomic stability and to verify whether the inhibition of p53 function in normal cells by pifithrin-alpha (PFT-alpha) may contribute in reducing the side effects of cancer therapy. Two human lymphoblastoid cell lines, derived from the same donor, TK6 (p53 wild type) and WTK1 (p53 mutated) have been treated with an anti-neoplastic drug, the etoposide (VP16), an inhibitor of DNA topoisomerase II in presence or in absence of the p53 inhibitor PFT-alpha. Following treatments with VP16 on TK6 and WTK1, we observed a higher induction of chromosome aberrations in WTK1 (p53 mutated) and of apoptosis in TK6 (p53 wild-type) cells. The p53 inhibition by PFT-alpha in VP16 treated TK6 cells produced an increase of chromosomal aberrations and a reduction of apoptosis. Therefore, the temporary suppression of the function of p53 by PFT-alpha, increasing the survival of the normal cells, could be a promising approach to reduce the side-effects of cancer therapy but it is important to consider that the surviving cells could be genetically modified and consequently the risk of secondary tumours could be increased.     

Reduced viability of human vascular endothelial cells cultured on Matrigel™

Optimal vascular homeostasis requires efficient control of both proliferation and elimination of vascular endothelial cells. Programmed cell death, or apoptosis, is the main mechanism controlling cell elimination, and it is an essential component of vascular formation. Human vascular endothelial cells die in vitro, if prevented from obligatory survival factors like growth factors or attachment and cell spreading, but very little is known about the mechanisms controlling endothelial cell elimination. Signaling from the extracellular matrix affects the behavior and functions of human umbilical vein endothelial cells (HUVECs), and we have recently demonstrated the beneficial effects of plating on the reconstituted extracellular matrix Matrigel™, on the inducible nitric oxide production of freshly isolated HUVECs. In this work we observed that cultured HUVECs formed typical capillary-like structures on Matrigel, but unexpectedly, after 24–48 hours their viability was gradually lost. Viability was measured with an assay based on mitochondrial reduction of reagent XTT. No decrease in viability was seen in freshly isolated HUVECs or in cultured fibroblasts during this time. It is known that cells often turn into apoptosis if they receive conflicting information from their surroundings, and apparently signaling from Matrigel to HUVECs, while at their in vitro proliferating phenotype, resulted in launching of the apoptotic machinery. Thus, proliferating and differentiated phenotypes of endothelial cells seemed to have different sensitivity to signals that induce apoptosis. J. Cell. Physiol. 176:92–98, 1998. © 1998 Wiley-Liss, Inc.     

Proteasome activation as a critical event of thymocyte apoptosis

Caspase activation may occur in a direct fashion as a result of CD95 death receptor crosslinking (exogenous pathway) or may be triggered indirectly, via a Bcl-2 inhibitable mitochondrial permeabilization event (endogenous pathway). Thymocyte apoptosis is generally accompanied by proteasome activation. If death is induced by DNA damage, inactivation of p53, overexpression of a Bcl-2 transgene, inhibition of protein synthesis, and antioxidants (N-acetylcyteine, catalase) prevent proteasome activation. Glucocorticoid-induced proteasome activation follows a similar pattern of inhibition except for p53. Caspase inhibition fails to affect proteasome activation induced by topoisomerase inhibition or glucocorticoid receptor ligation. In contrast, caspase activation (but not p53 knockout or Bcl-2 overexpression) does interfere with proteasome activation induced by CD95. Specific inhibition of proteasomes with lactacystin or MG123 blocks caspase activation at a pre-mitochondrial level if thymocyte apoptosis is induced by DNA damage or glucocorticoids. In strict contrast, proteasome inhibition has no inhibitory effect on the mitochondrial and nuclear phases of apoptosis induced via CD95. Thus, proteasome activation is a critical event of thymocyte apoptosis stimulated via the endogenous pathway yet dispensable for CD95-triggered death.     

Pifithrin-alpha protects against doxorubicin-induced apoptosis and acute cardiotoxicity in mice

The present experiments were designed to evaluate the effects of pifithrin-alpha (PFT-alpha), which is a p53 inhibitor, on doxorubicin (DOX)-induced apoptosis and cardiac injury. Administration of DOX (22.5 mg/kg ip) in mice upregulated the mRNA levels of Bax and MDM2, whereas PFT-alpha attenuated those levels when administered at a total dose of 4.4 mg/kg at 30 min before and 3 h after DOX challenge. DOX treatment led to an upregulation of p53 protein levels, which was preceded by elevated levels of phosphorylated p53 at Ser15. PFT-alpha had no effect on the level of p53 or its phosphorylated form. The protein levels of Bax and MDM2 were elevated by DOX and attenuated by PFT-alpha. DOX gave rise to increased apoptosis-positive nuclei in cardiac cells, elevated serum creatine phosphokinase, ultrastructural alterations, and cardiac dysfunction. PFT-alpha offered protection against all of the aforementioned changes. Finally, PFT-alpha did not interfere with the antitumor potency of DOX. This study demonstrates that PFT-alpha effectively inhibits DOX-induced cardiomyocyte apoptosis, which suggests that PFT-alpha has the potential to protect cancer patients against DOX-induced cardiac injury.     

Contribution of p16INK4a and p21CIP1 pathways to induction of premature senescence of human endothelial cells: permissive role of p53

We have previously found that nonenzymatically glycated collagen I (GC), mimicking diabetic microenvironment, can induce senescent phenotype in early passage human umbilical vein endothelial cells (HUVECs). In the present study, we explored the functional involvement of cell cycle checkpoint pathways in initiating GC-induced premature endothelial cell senescence. When compared with native collagen, early passage HUVECs showed increased p53, p21(CIP1) (p21), and p16(INK4a) (p16) mRNA expression after exposure to GC. Twenty-four hours after transfection of p16, p21, and p53-enhanced green fluorescent protein (EGFP) recombinant plasmids, HUVECs entered G(1)-phase cell cycle arrest. By days 3 and 5, HUVECs transfected with p16-EGFP showed an increased proportion of senescent cells, and this increase was more prominent in the GFP-positive cell population, which exhibited 68% of senescent cells. Transfection of p21 also induced senescence but only by day 5. Cotransfection of p16 and p21 showed no additive effect. Transfection of p21 or p53 induced apoptosis in HUVECs. Next, we suppressed endogenous p53, p21, p16, or retinoblastoma (Rb) gene expression through small interference RNA strategy and investigated their influence in p16- and p21-initiated endothelial cell senescence. Analysis indicated that suppression of p53 expression can abolish senescence induced by p16 overexpression. Paradoxically, this effect was not observed when p21 was suppressed. On the other hand, suppression of Rb eliminated senescence initiated by either p16 or p21 overexpression. In summary, the p53/p21 pathway is mainly responsible for GC-induced apoptosis, but the coordinated activation of the p53/p21 and p16 pathway is responsible for GC-induced endothelial cell senescence through a Rb-dependent mechanism.     

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.     

AREL1 resists the apoptosis induced by TGF-β by inhibiting SMAC in vascular endothelial cells

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-β (TGF-β), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-β treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-β. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-β treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-β induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.     

Progesterone receptor activation of extranuclear signaling pathways in regulating p53 expression in vascular endothelial cells

We previously showed that progesterone (P4) inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) through a p53-dependent pathway. Now we investigated further the molecular mechanism underlying the hormone activity. In cultured HUVECs, P4 increased the protein levels of phosphorylated Src (p-Src), Raf-1, and ERK. The levels of p-Src and p-Src-progesterone receptor complex in HUVECs were increased by P4 treatment. These effects were blocked by pretreatment with a progesterone receptor antagonist, RU486. The P4-induced increase in p53 transactivity was abolished by pretreatment with Src kinase inhibitors. Moreover, administration with cSrc antisense oligonucleotide prevented the P4-induced increases of the levels of p53 mRNA and protein. These data suggest that P4-induced up-regulation of p53 might be mediated through activation of cSrc. Pretreatment with Src kinase inhibitors also prevented P4-induced membrane translocation of Kras and increases of the protein levels of phosphorylated Raf and phosphorylated ERK. Transfection with dominant-negative ERK2 prevented the P4-induced increases of protein level and promoter activity of p53 and a decrease of thymidine incorporation. P4 also increased nuclear factor-κB (NF-κB) nuclear translocation and NF-κB binding onto the p53 promoter. These effects were abolished by pretreatment with ERK inhibitors. The P4-induced up-regulation of the p53 promoter activity was prevented by preadministration with dominant-negative ERK2 or NF-κB inhibitors. Taken together, our data suggest that the cSrc/Kras/Raf-1/ERK2/NF-κB signaling pathway contributes to the P4-induced up-regulation of p53 in HUVECs. These findings highlight progesterone receptor activation of extranuclear signaling pathways in regulating p53 and cell cycle progression in HUVECs.     

Monocyte chemoattractant protein-1 induces endothelial cell apoptosis in vitro through a p53-dependent mitochondrial pathway

The cystine-cystine (CC) chemokine monocyte chemoattractant protein-1 (MCP-1) has been established playing a pathogenic role in the development of atherosclerosis due to its chemotactic ability of leading monocytes to locate to subendothelia. Recent studies have revealed more MCP-1 functions other than chemotaxis. Here we reported that various concentrations (0.1-100 ng/ml) of MCP-1 induced human umbilical vein endothelial cell (HUVEC) strain CRL-1730 apoptosis, caspase-9 activation, and a couple of mitochondrial alterations. Moreover, MCP-1 upregulated p53 expression of HUVECs and the p53-specific inhibitor pifithrin-α (PFTα) rescued the MCP-1-induced apoptosis of HUVECs. Furthermore, PKC (protein kinase C) activation or inhibition might also affect HUVECs apoptosis induced by MCP-1. These findings together demonstrate that MCP-1 exerts direct proapoptotic effects on HUVECs in vitro via a p53-dependent mitochondrial pathway.     

皮肤血管瘤组织中WT-1、Bcl-2、P53的表达及意义

目的研究肾母细胞瘤基因（WT-1）、Bcl-2和P53在增生期、退化期血管瘤及正常组织中的表达,探讨其意义及相互关联。方法采用免疫组化SP法检测人皮肤血管瘤组织中WT1、Bcl-2和P53在增生期、退化期及正常皮肤组织中血管内皮细胞中的表达水平,利用计算机成像分析技术检测不同时期皮肤血管瘤与正常皮肤组织WT1、Bcl-2和P53的平均光密度及其阳性面积率。结果1.WT-1在退化期血管瘤中有较强表达,而在增生期血管瘤和正常皮肤组织中表达微弱或不表达（P〈0.05）。2.Bcl-2在增生期血管瘤的表达明显高于退化期血管瘤和正常皮肤组织（P〈0.01）;Bcl-2在退化期血管瘤的表达与正常皮肤组织相比,差异无显著性（P〉0.05）。3.p53基因在增生期血管瘤组织中表达水平高于退化期,差异有极显著性意义（P〈0.01）,退化期血管瘤p53基因表达水平与正常皮肤组织相比,差异无显著性意义（P〉0.05）。结论1.WT-1可能通过促进内皮细胞凋亡而抑制血管瘤的增生;2.Bcl-2可能是通过抑制内皮细胞的凋亡,使其增殖和凋亡失衡;3.P53可能促进了血管瘤增生期内皮细胞的增殖,使血管内皮细胞大量生成。