Toll-like Receptor 4 Is Expressed on Intestinal Stem Cells and Regulates Their Proliferation and Apoptosis via the p53 Up-regulated Modulator of Apoptosis

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.     

Hyperglycemia induces apoptosis of pancreatic islet endothelial cells via reactive nitrogen species-mediated Jun N-terminal kinase activation

Hyperglycemia significantly stimulates pancreatic islet endothelial cell apoptosis; however, the precise mechanisms are not fully understood. In the present study, treating pancreatic islet endothelial (MS-1) cells with high glucose (30 mmol/l) but not mannitol significantly increased the number of apoptotic cells as compared with a physiological glucose concentration (5.5 mmol/l). Hyperglycemia significantly stimulated the expression of inducible nitric oxide synthase (iNOS) and production of NO and peroxynitrite (ONOO⁻), relevant to MS-1 cell apoptosis. Moreover, induced reactive nitrogen species (RNS) significantly increased the expression of bax, cleaved caspase-3 and poly adenosine diphosphate (ADP)-ribose polymerase (PARP) via JNK activation, but the expression of bcl-2 was not altered. Furthermore, SP600125 (a specific inhibitor of JNK) and 1400W (a specific inhibitor of iNOS) significantly attenuated cell apoptosis induced by high glucose. Therefore, hyperglycemia triggers MS-1 cell apoptosis by activating an intrinsic-dependent apoptotic pathway via RNS-mediated JNK activation.     

熊去氧胆酸对阿霉素诱导的H9c2心肌细胞的影响及机制研究

目的:探讨熊去氧胆酸(UDCA)对阿霉素(DOX)诱导的H9c2心肌细胞损伤的影响及机制.方法:体外培养H9c2细胞,1 μMDOX和不同浓度UDCA处理H9c2,CCK-8法测定细胞活力;实时定量聚合酶链反应检测心肌细胞凋亡分子Bax及炎症因子IL-1β、IL-6的表达;Western blotting检测UDCA对...     

Synthesis and proapoptotic activity of oleanolic acid derived amides

Thirty-one different 3-O-acetyl-OA derived amides have been prepared and screened for their cytotoxic activity. In the SRB assays nearly all the carboxamides displayed good cytotoxicity in the low μM range for several human tumor cell lines. Low EC₅₀ values were obtained especially for the picolinylamides 14–16, for a N-[2-(dimethylamino)-ethyl] derivative 27 and a N-[2-(pyrrolinyl)-ethyl] carboxamide 28. These compounds were submitted to an extensive biological testing and proved compound 15 to act mainly by an arrest of the tumor cells in the S phase of the cell cycle. Cell death occurred by autophagy while compounds 27 and 28 triggered apoptosis.     

Human and Pneumococcal Cell Surface Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) Proteins Are Both Ligands of Human C1q Protein

C1q, a key component of the classical complement pathway, is a major player in the response to microbial infection and has been shown to detect noxious altered-self substances such as apoptotic cells. In this work, using complementary experimental approaches, we identified the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a C1q partner when exposed at the surface of human pathogenic bacteria Streptococcus pneumoniae and human apoptotic cells. The membrane-associated GAPDH on HeLa cells bound the globular regions of C1q as demonstrated by pulldown and cell surface co-localization experiments. Pneumococcal strains deficient in surface-exposed GAPDH harbored a decreased level of C1q recognition when compared with the wild-type strains. Both recombinant human and pneumococcal GAPDHs interacted avidly with C1q as measured by surface plasmon resonance experiments (K_D = 0.34–2.17 nm). In addition, GAPDH-C1q complexes were observed by transmission electron microscopy after cross-linking. The purified pneumococcal GAPDH protein activated C1 in an in vitro assay unlike the human form. Deposition of C1q, C3b, and C4b from human serum at the surface of pneumococcal cells was dependent on the presence of surface-exposed GAPDH. This ability of C1q to sense both human and bacterial GAPDHs sheds new insights on the role of this important defense collagen molecule in modulating the immune response.     

Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells

Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis.     

Analysis of a novel mechanism of neuronal toxicity produced by an apolipoprotein E-derived peptide

The apolipoprotein E (apoE)-derived peptide (141-155)2 has a neurotoxic effect, implying that apoE itself could be a source of toxicity in Alzheimer's disease brain. We characterized the toxicity of this peptide on superior cervical ganglion (SCG) neurons and compared the death with the apoptotic death that occurs after nerve growth factor (NGF) deprivation in these cells. A dose of 10 microM apoE (141-155)2 resulted in the death of approximately 50% of the neurons within 24 h. Nuclear condensation and DNA fragmentation preceded the death. However, most inhibitors of NGF deprivation-induced death, including the caspase inhibitor Boc-aspartyl(O-methyl)fluoromethyl ketone and genetic deletion of bax-/-, had no effect on the toxicity. Inclusion of depolarizing levels of potassium did block the toxicity. Receptor-associated peptide (RAP), an antagonist for apoE receptors, did not protect cells in either SCG or hippocampal cultures. In addition, RAP had no effect on internalization of the apoE peptide. These data support the observation that apoE (141-155)2 is neurotoxic but suggest that the neurotoxicity is distinct from classical apoptosis or necrosis. Furthermore, these results indicate that the toxic effect may occur independently of members of the low-density lipoprotein receptor gene family.     

Apoptosis induced by (di-isopropyloxyphoryl-Trp)2-Lys-OCH3 in K562 and HeLa cells

According to the method used in our laboratory, our group synthesized (DIPP-Trp)₂-Lys-OCH₃. It inhibited the proliferation of K562 and HeLa cells in a dose-and time-dependent manner with an IC₅₀ of 15.12 and 42.23 µM, respectively. (DIPP-Trp)₂-Lys-OCH₃ induced a dose-dependent increase of the G2/M cell population in K562 cells, and S cell population in HeLa cells; the sub-G0 population increased dramatically in both cell lines as seen by PI staining experiments using a FACS Calibur Flow cytometer (BeckmanCoulter, USA). Phosphatidylserine could significantly translocate to the surface of the membrane in (DIPP-Trp)₂-Lys-OCH₃-treated K562 and HeLa cells. The increase of an early apoptotic population was observed in a dose-dependent manner by both annexin-FITC and PI staining. It was concluded that (DIPP-Trp)₂-Lys-OCH₃ not only induced cells to enter into apoptosis, but also affected the progress of the cell cycle. It may have arrested the K562 and HeLa cells in the G₂/M, S phases, respectively. The apoptotic pathway was pulsed at this point, resulting in the treated cells entering into programmed cell death. (DIPP-Trp)₂-Lys-OCH₃ is a potential anticancer drug that intervenes in the signalling pathway.     

葡萄糖和胰岛素对血管内皮细胞中microRNA-21的表达影响

目的:研究miRNA-21和程序性细胞死亡因子4(PDCD4)在不同浓度葡萄糖或胰岛素培养血管内皮细胞中的表达情况,探讨miRNA-21对内皮细胞凋亡功能的影响。方法:用不同浓度D-葡萄糖(5.5、11、22及33mM)和胰岛素(0、1、10、50、100及1000nM)分别干预人脐静脉内皮细胞(HUVECs),2天后收集细胞用实时荧光定量RT-PCR方法检测细胞中miRNA-21及PDCD4的mRNA表达情况,用蛋白印迹方法检测PDCD4在细胞中的表达变化。结果:与5.5mM葡萄糖对照组相比,高葡萄糖浓度组(11、22及33mM)中HUVECs的miRNA-21随着葡萄糖浓度的增加表达明显降低(p<0.01);PDCD4的蛋白表达增加(p<0.05),但PDCD4的mRNA表达无差异(p>0.05)。与0nM胰岛素组相比,10、50、100、1000nM胰岛素组细胞中miRNA-21表达下降(p<0.05),PDCD4的蛋白表达增加(p<0.01),1000nM组miRNA-21表达降低程度和PDCD4的蛋白表达增加程度最为明显(p<0.05),但不同浓度胰岛素对PDCD4的mRNA表达无差异(p>0.05...     

Alpha-tocopheryl succinate sensitizes human colon cancer cells to exisulind-induced apoptosis

Sulindac sulfone (also known as exisulind) and its chemical derivatives are promising anticancer agents capable of inducing apoptosis in a variety of malignant cell types with minimal toxicity to normal cells. Here, we tested the ability of alpha-tocopheryl succinate (TOS), another promising anticancer agent, to sensitize colon cancer cells to exisulind-induced apoptosis. We found that sub-apoptotic doses of TOS greatly enhanced exisulind-induced growth suppression and apoptosis in the HCT116, LoVo and SNU-C4 human colon cancer cell lines. Our results revealed that this was accounted for primarily by an augmented cleavage of poly(ADP-ribose) polymerase (PARP) and enhanced activation of caspase-8, -9 and -3. Pretreatment with z-VAD-FMK (a pan-caspase inhibitor), z-IETD-FMK (a caspase-8 inhibitor) or z-LEHD-FMK (a caspase-9 inhibitor) blocked TOS and exisulind cotreatment-induced PARP cleavage and apoptosis. Furthermore, TOS/exisulind cotreatment induced JNK phosphorylation, while pretreatment with SP600151 (a JNK inhibitor) partially blocked cotreatment-induced caspase-dependent PARP cleavage and apoptosis. Taken together, these findings indicate that TOS sensitizes human colon cancer cells to exisulind-induced apoptosis. Apoptotic synergy induced by exisulind plus TOS seems likely to be mediated through a mechanism involving activation of caspases and JNK. S.-J. Lim, Y.-J. Lee both authors are contributed equally to this study.