依托度酸诱导SMMC7721细胞凋亡的分子机理研究

目的：探讨选择性环氧合酶抑制剂依托度酸（etodolac）诱导肝癌SMMC7721细胞凋亡的分子机理。 方法： 采用流式细胞术、DNA琼脂糖凝胶电泳法测定细胞凋亡情况；Western blotting法检测不同浓度etodolac处理后凋亡相关蛋白Bcl-2、Bax表达的变化；流式细胞术检测半胱氨酸酶-3 (caspase-3)活性的变化；TransAMTM NF-κB p65/p50核转录因子活性检测试剂盒检测核因子-κB (NF-κB)活性变化。 结果： 流式细胞术显示etodolac（0.25、0.50、1.0、2.0 mmol/L）作用SMMC7721细胞48 h后，与对照组（0 mmol/L）相比，出现明显凋亡峰（P<0.01 vs control）；高浓度etodolac处理后DNA琼脂糖凝胶电泳出现明显的DNA Ladder, 凋亡相关蛋白Bcl-2表达下降，Bax表达增加；与对照组相比，低浓度组（0.25 mmol/L）caspase-3活性未明显活化（P>0.05），NF-κB活性也未受明显抑制（P>0.05），随着etodolac浓度的增大（0.50、1.0、2.0 mmol/L），caspase-3活性明显活化（P<0.05 vs control）； NF-κB活性明显受到抑制（P<0.05 vs control）。经Pearson 相关分析，caspase-3活性和NF-κB活性呈显著负相关（r=0.919, P<0.01）。 结论： 选择性COX-2抑制剂etodolac可能通过抑制NF-κB结合活性，调节Bcl-2、Bax蛋白表达，活化caspase-3，从而诱导肝癌SMMC7721细胞凋亡。     

Influence of infliximab on keratinocyte apoptosis in psoriasis patients

Biological drugs targeting tumor necrosis factor-α, such as infliximab, are highly effective in psoriasis. The interference with keratinocyte apoptosis has been included among the possible effects of infliximab in psoriasis, although the available data are still controversial. The purpose of our study was to verify the action of infliximab on psoriatic keratinocytes. Keratinocyte apoptosis was evaluated in the lesional psoriatic skin of 11 patients at baseline and a different time point during treatment with infliximab. Infliximab (5?mg/kg) was given intravenously at weeks 0, 2, and 6, followed by maintenance infusions every 8 weeks. Pretreatment with intravenous hydrocortisone was performed prior to each infusion. Keratinocytes with apoptotic features were histologically identified according to the following changes: chromatin condensation at the periphery of the nucleus, cytoplasmic vesiculation, nuclear fragmentation, nuclear pyknosis. Immunohistochemical assessment of p53 and caspase-3 expression was also performed. At baseline, prior to treatment with infliximab, lesional epidermis showed 1.2–3.2% p53-positive apoptotic keratinocytes in the basal zone. The number of p53-positive apoptotic keratinocytes increased after treatment with infliximab, already at day 1–2 after the first infusion, and such cells were localized at basal and suprabasal layers or were through all layers. There was no immunoreactivity for caspase-3 at any time point examined. Our results suggest that induction of p53-related keratinocyte apoptosis might be one of the mechanisms of infliximab action in psoriasis.     

Targeting keratinocyte apoptosis in the treatment of atopic dermatitis and allergic contact dermatitis.

BACKGROUND: Activation and skin-selective homing of T cells and effector functions in the skin represent sequential events in the pathogenesis of atopic dermatitis and allergic contact dermatitis. OBJECTIVE: T cell-mediated keratinocyte apoptosis plays a key pathogenetic role in the formation of eczematous dermatitis. IFN-gamma released from activated T cells upregulates Fas on ke-ratinocytes, which renders them susceptible to apoptosis. The lethal hit is given to keratinocytes by means of Fas ligand expressed on the T-cell surface or released to the inflammatory microenvironment. We sought to investigate whether drugs used for the treatment of eczematous disorders interfere with this pathogenic pathway. METHODS: T cell-mediated, Fas-induced keratinocyte apoptosis in a keratinocyte-T cell coculture system serves as an in vitro model of eczematous dermatitis. We tested, in this model, whether immunomodulatory agents (dexamethasone, cyclosporine A, rapamycine, tacrolimus/FK506, intravenous immunoglobulin [IVIG], and theophylline) are able to inhibit apoptosis of keratinocytes. Additionally, skin biopsy specimens from patients with untreated and successfully treated eczematous dermatitis were evaluated for keratinocyte apoptosis. RESULTS: Dexamethasone, cyclosporine A, FK506, rapamycine, and IVIG are inhibitors of keratinocyte apoptosis induced by activated T cells. This effect is mediated by 2 major mechanisms directed on T cells or keratinocytes. T-cell activation was mainly inhibited by dexamethasone, FK506, cyclosporine A, and rapamycine. Interestingly, high-dose dexamethasone and IVIG directly inhibited Fas-mediated keratinocyte apoptosis. In vivo keratinocyte apoptosis was significantly reduced after successful topical treatment of eczematous lesions. CONCLUSION: These results demonstrate mechanisms of action of current treatment approaches and provide a future for more focused therapeutic applications.     

T-2 toxin-induced apoptosis in rat keratinocyte primary cultures

T-2 toxin, a kind of trichothecene mycotoxins produced by the genus Fusarium, induces apoptosis in basal keratinocytes when topically applied to the dorsal skin of rats. In the present study, direct effects of T-2 toxin on keratinocyte primary cultures obtained from newborn rats were examined after the third passage. Keratinocyte medium containing 0.25 microg/ml of T-2 toxin dissolved in dimethyl sulfoxide or solvent alone was added to 4-day cultures and incubated at 37 degrees C. At 0.5, 1, 3, 5, 7, and 9 h after treatment (h), feeder layer was separated from flasks, and cells were trypsinized. Cell viability was estimated by trypan blue exclusion method. In addition, RNA was obtained and RT-PCR was performed. Samples obtained from slide cultures at 3, 6, 9, and 12 h were fixed in 4% paraformaldehyde or 2.5% glutaraldehyde for morphological examination. After T-2 toxin application, cell viability decreased to 40% at 3 h. At 6 h, small-sized keratinocytes showed pyknosis or karyorrhexis, resulting in detachment from slides. The number of such cells increased until 12 h. These small-sized keratinocytes showed ultrastructural changes characteristic for apoptosis. At the same time, large squamous keratinocytes showed intracytoplasmic edema. The expression of apoptosis-related genes (c-fos and c-jun) and cytokines (TNF-alpha and IL-1beta) mRNAs markedly increased before the development of apoptosis. These findings indicate that c-fos and c-jun oncogenes and TNF-alpha and IL-1beta play an important role in the development of T-2 toxin-induced apoptosis in keratinocytes.     

The mechanism for keratinocyte detaching from pH-responsive chitosan

In this study, we compared the detachment ratio of HaCaT and Hs68 cells from pH-responsive chitosan surface by raising medium pH from 7.20 to 7.65 for 60 min. The detachment ratio of elongated Hs68 cells was over 75%, but that of round-shaped HaCaT cells was less than 50%, even extending the incubation time to 6 h or enhancing the cytoskeletal contractile force with the Rho activator CN01. However, the addition of 2 mm of EDTA into the medium at pH 7.65 could effectively detach HaCaT cells (detachment ratio > 90%), indicating that the calcium ion played an important role in the detachment process. Therefore, the family of Ca⁺²-dependent integrin receptors was examined by RT-PCR, real-time PCR and immunocytochemistry. It was found the expression of integrin β4 (ITGb4) was HaCaT cell-specific and the mRNA level of ITGb4 in undetached HaCaT cells was significantly higher than that in detached ones. By modulating ITGb4 activity with specific functional blocking antibody ASC-8, the detachment ratio of HaCaT cells could be increased to be greater than 85%. Conversely, the addition of the ligand of ITGb4 laminin into the culture system decreased the medium pH-induced detachment ratio for HaCaT cells, but not for Hs68 cells. Further addition of ASC-8 could rescue the effect of laminin on preventing the detachment of HaCaT cells from pH-sensitive chitosan surface. Therefore, this study demonstrated the interaction of ITGb4 and laminin played an important role in controlling the detachment of HaCaT cells on pH-responsive chitosan.     

Methotrexate treatment provokes apoptosis of proliferating keratinocyte in psoriasis patients

Psoriasis is a chronic inflammatory skin disease characterized by hyper proliferation of keratinocytes. Recent data show that the epidermis thickening in psoriasis may be related to imbalance of homeostasis caused by abnormal apoptotic process. Maintenance of keratinocyte apoptotic process is very important in psoriasis. Methotrexate (MTX) has been used for many years to restore the normal skin in psoriasis condition. However, the exact mechanism of MTX in psoriasis condition is poorly understood. The aim of this study was to examine the role of MTX on keratinocyte apoptosis pathway in psoriasis patients. A total of 58 psoriasis vulgaris patients were recruited for this study. Nonlesional skin biopsies served as control. Skin biopsies of psoriatic patients were collected and analyzed for cytosolic, mitochondria and total cytochrome c by ELISA. Expression of caspase-9, NFκBp65, pAkt1 by western blot, real-time PCR and immunohistochemical analysis of c-FLIP protein was analyzed in nonlesional and lesional skin biopsies before (day 0) and after (at the end of 6 and 12 weeks) MTX treatment. After MTX treatment, a significant increase in cytochrome c was observed when compared with before MTX treatment in psoriasis patients (p < 0.001). Protein and gene expression of cleaved caspase-9 were significantly increased after MTX treatment, whereas the expression of Bcl-xL, c-FLIP, NFκBp65, pAkt1 significantly downregulated after MTX treatment. In conclusion, these results showed that intrinsic apoptotic pathway induced by MTX eventually adds the beneficial therapeutic role of MTX in psoriasis by controlling the acanthosis.     

Molecular aspects of apoptosis

Corresponding to its importance in cell count homeostasis in the body, apoptosis is a tightly regulated phenomenon. Both extracellular and intracellular molecules provide multiple regulatory and counter-regulatory pathways. Cell death is usually a response to the cell microenvironment, where the absence of certain factors (survival factors) or the presence of lethal factors promotes apoptosis. Surrounding cells, soluble mediators and the extracellular matrix regulate cell death and survival. Surrounding cells can synthesize survival or lethal factors. The intracellular regulation of apoptosis is also one of the forefront fields in biomedicine research. During the past five years, tremendous progress has been made in understanding apoptosis as a result of molecular identification of the key components of this intracellular suicide program. Biochemical activation of these key components of the cell death program is responsible for the morphological changes observed in apoptosis, including mitochondrial damage, nuclear membrane breakdown, DNA fragmentation, chromatin condensation and the formation of apoptotic bodies. Caspase activation plays a central role in the execution of apoptosis. Most caspases are constitutively expressed as inactive proenzymes (procaspases) in the cytosol and according to some reports in the mitohondria. Caspases are sequentially activated by proteolysis during apoptosis. In this review, we focus on the biochemical pathways that control caspase activation, particularly the activation pathways that are initiated by cell surface death receptors and mitochondria.     

Molecular mechanism of SSFA2 deletion inhibiting cell proliferation and promoting cell apoptosis in glioma

Gliomas are the most common primary brain malignant tumors in humans. Glioblastoma multiforme(GBM) is the most malignant intracranial tumor with a relatively poor prognosis. There promote us to find effective anti-cancer therapies to reduce cancer mortality. By using bioinformatic analysis, we found SSFA2 as a gene with elevated expression in the glioma tissues. We detected the expression of SSFA2 in glioma tissues and in the glioma cell lines, as well as in normal brain tissues. SSFA2 expression was higher in glioma tissues, especially in glioblastoma multiforme than normal brain tissues. Subsequently, we found that down-regulate SSFA2 in glioma cell lines can regulate the cell cycle to reduce the proliferation ability and induce the early apoptosis rate in shSSFA2 cells relative to control cells. Moreover, we found that down-regulate SSFA2 in glioma cell line U87(shSSFA2-U87) inhibited the growth effectiveness compared to the control cell line U87. These result reveals us that SSFA2 may act as oncogene to promote the progression of glioma. For further research specific mechanisms of SSFA2 in gliomas, we used the gene chip to detect the downstream gene in U87. We found that 30 genes also may be as target gene of SSFA2, and we testify the protein expression by western-blot. The result reveal that IL1A, IL1B and CDK6 as target gene of SSFA2 to regulate the progression of glioma. These finding suggest that SSFA2 could be a new therapeutic target for gliomas.     

Molecular mechanisms of apoptosis pathology

Apoptosis--a programmed cell death--is a general biological mechanism responsible for maintenance of a permanent cell number. Impairment of apoptosis regulation entails disorders in homeostasis and various diseases. This explains biological and medical importance of apoptosis. Molecular grounds of apoptosis regulation, physiological regulation depending on the cell type, examples of apoptosis disregulation leading to definite diseases, approaches to treatment of some diseases basing on knowledge of apoptosis regulation are provided.     

P38丝裂原活化蛋白激酶在紫外线诱导角质形成细胞凋亡中的作用

目的 探讨中波紫外线(UVB)诱导角质形成细胞凋亡机制.方法 使用0、20、30、40、60、70、90 mJ/cm2 UVB辐射人角质形成细胞(HaCaT),辐射后0、1、2、3、4、6、24、48 h收集细胞和上清.流式细胞仪检测细胞周期和凋亡;ELISA检测上清液中TNF-α水平;Western blot检测P38丝裂原活化蛋白激酶(P38MAPK)和磷酸化P38丝裂原活化蛋白激酶(p-P38MAPK)的表达.结果 UVB(30 mJ/cm2)辐射后,HaCaT细胞分泌TNF-α逐渐增加,24 h达高峰;P38抑制剂SB203580完全抑制TNF-α的分泌,部分阻止了细胞凋亡;以不同剂量UVB辐射HacaT细胞,在30 mJ/cm2时p-P38 MAPK表达量最高,且辐射后即可检测到p-P38 MAPK的表达,1 h时达高峰,以后逐渐下降,而P38MAPK表达量没有明显变化.结论 紫外线可通过激活P38 MAPK途经,促进TNF-α分泌来诱导角质形成细胞凋亡.     

凋亡的双向调节机制

细胞凋亡的基本过程大致包括刺激期、信号传递期、效应期 (人类效应物半胱氨酸蛋白酶类总称ｃａｓｐａｓｅｓ)及死后期。在从线虫到人类不同有机体细胞凋亡的前几期研究中逐步认识到 ,凋亡的发生是细胞受促进性和抑制性双向因素共同调节的结果 ,已发现凋亡的双向调节机理分述如下。一、含死亡区分子的作用在凋亡的刺激期 ,细胞接受的刺激因素包括各种细胞因子如肿瘤坏死因子 (ＴＮＦ)、ＦａｓＬ、干扰素 (ＩＦＮ) γ、白细胞介素(ＩＬ) 6 ,某些生长因子如神经生长因子 (ＮＧＦ)、内皮生长因子(ＥＧＦ)、血小板源生长因子 (ＰＤＧＦ)等 ,这…     

Resistance of CD1d-/- mice to ultraviolet-induced skin cancer is associated with increased apoptosis

Inhibition of p53-induced epidermal apoptosis, generation of p53 mutations, and suppressor T cells are the critical events responsible for the induction and development of UV-induced skin cancers. Recently, we demonstrated that CD1d knockout mice were resistant to UV-induced immunosuppression, prompting us to further address the role of CD1d in regulating UV carcinogenesis. We, therefore, investigated the response of wild-type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. Surprisingly, keratinocytes and fibroblasts from CD1d-/- mice were more sensitive to UV-induced apoptosis and persisted longer than cells derived from WT mice. In addition, epidermis and dermis taken from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. Our findings identify an unexpected and novel function for CD1d as a critical molecule regulating UV carcinogenesis, by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts.     

颗粒酶A介导的凋亡机制研究现状

目的:CTL细胞杀伤靶细胞有两个重要方式:即分泌型和非分泌型杀伤。前者指的是如穿孔素/(颗)粒酶类介质使靶细胞裂解,后者主要指通过FAS-FASL结合等一类途径诱导凋亡。(颗)粒酶A是(颗)粒酶家族中含量较多、作用方式较独特、且相当重要的一种物质。穿孔素/(颗)粒酶A/SET复合物介导的凋亡途径是一条重要的途径,它通过引起单股DNA链缺刻和DNA修复障碍导致细胞凋亡。其在杀伤肿瘤细胞、病毒感染细胞及异体移植物细胞有重要意义,近年来对穿孔素/(颗)粒酶A/SET复合物介导的细胞凋亡途径研究比较多,本文拟在这方面作一些综述。     

Massive apoptosis in infantile myofibromatosis. A putative mechanism of tumor regression.

Two cases of solitary infantile myofibromatosis (IM) are presented. Solitary IM are tumors prone to spontaneous regression. Histopathologically, several tumor lobules in our IM cases had central areas of massive cell death, with nuclear pyknosis, cytoplasmic hyalinization and nuclear fragmentation but without lymphoid or neutrophilic cell infiltration. These central cell death areas consisted of about 40% in case 2 and 50% in case 1 of the entire tumor tissues, respectively. Electron microscopy revealed that the condensed nuclei and cytoplasm were fragmented into "apoptotic bodies", with or without phagocytosis by histiocytes. DNA fragmentation, as evidenced by the terminal deoxy transferase-mediated uptake of biotinylated dUTP, was identified at massive cell death areas on paraffin sections from both cases. A characteristic 180- to 190-bp nucleosomal ladder was detected in DNA obtained from the tumor cells in case 1. The collective evidence suggested that these tumors underwent a central, massive apoptosis. As massive cell death similar to that seen in the present cases has been described in other documented cases of IM, we propose that the spontaneous regression that frequently occurs with this type of tumor may be mediated by massive apoptotic cell death.     

Molecular mechanism of troponin-C function

Conclusions There is now a large body of evidence in support of the view that Ca²⁺ binding to the low affinity sites of TnC induces a movement of helices B and C away from helices A and D, thus opening a hydrophobic cavity, the site of interaction with TnI. Another site of similar structure is formed by the helical segments in the C-terminal domain. Both sites appear to interact with the inhibitory segment of TnI. Whereas the interactions at both sites are necessary for the full regulatory activity of TnC, the interaction at the C-terminal domain stabilizes the complex and that involving the N-terminal domain is directly linked to the release of inhibition. In the absence of Ca²⁺ the inhibitory region of TnI would preferentially bind to actin and on Ca²⁺ binding to sites I and II it would switch to the site in the N-terminal domain of TnC. Detachment of TnI from actin gives rise to further events in thin filament regulation.     

Molecular mechanism of paramyxovirus budding

Components of paramyxoviruses are assembled at the plasma membrane of infected cells, and progeny viruses are formed by the budding process. Although the molecular mechanisms that drive budding (membrane curving and "pinching-off" reaction) are not well understood, the viral matrix (M) protein is thought to play a major role in the process. The M protein forms a dense layer tightly associated with the inner leaflet of the plasma membrane of infected cells. Expression of the M protein of some paramyxoviruses results in the formation and release of virus-like particles that contain the M protein; thus, in these viruses, the M protein alone can apparently trigger all steps required for the formation and release of virus-like particles. M also interacts specifically with viral envelope glycoproteins and nucleocapsids and is involved in directed transport of viral components to the budding site at the apical surface of polarized cells. In addition, protein-protein interactions between M and the cytoplasmic tail of viral glycoproteins and between M and the nucleocapsid affect the efficiency of virus production. The structural organization of the virion and the functions of the M protein clearly indicate that this protein orchestrates the budding of paramyxovirus.     

Molecular cytogenetic characterization of human papillomavirus16-transformed foreskin keratinocyte cell line 16-MT

Anogenital cancers are closely associated with human papillomavirus (HPV), and HPV-infected individuals, particularly those with high-grade dysplasias, are at increased risk for cervical and anal cancers. Although genomic instability has been documented in HPV-infected keratinocytes, the full spectrum of genetic changes in HPV-associated lesions has not been fully defined. To address this, we examined an HPV16-transformed foreskin keratinocyte cell line, 16-MT, by GTG-banding, spectral karyotyping (SKY), and array comparative genomic hybridization (array CGH); these analyses revealed multiple numerical, complex, and cryptic chromosome rearrangements. Based on GTG-banding, the 16-MT karyotype was interpreted as 78-83,XXY,+add(1)(p36.3),+3,+4,+5,+5,+7,+8,+i(8)(q10)x2,+10,?der(12),der(13;14)(q10;q10),+15,+16,add(19)(q13.3),+21,+21,-22[cp20]. Multicolor analysis by SKY confirmed and further characterized the anomalies identified by GTG banding. The add(1) was identified as a der(1)(1qter-->1q25::1p36.1-->1qter), the add(19) as a dup(19), and the der(12) interpreted as a der(11) involving a duplication of chromosome 11 material and rearrangement with chromosome 19. In addition, previously unidentified der(9)t(9;22), der(3)t(3;19), and der(4)t(4;9) were noted. The 16-MT cell line showed losses and gains of DNA due to unbalanced translocations and complex rearrangements of regions containing known tumor suppressor genes. Chromosomal changes in these regions might explain the increased risk of cancer associated with HPV. Also, array CGH detected copy-number gains or amplifications of chromosomes 2, 8, 10, and 11 and deletions of chromosomes 3, 4, 11, and 15. These results provide the basis for the identification of candidate oncogenes responsible for cervical and anal cancer in amplified regions, and for putative tumor suppressor genes in commonly deleted regions like 11q22-23. Furthermore, these data represent the first full characterization of the HPV-positive cell line 16-MT.