去乙酰化酶抑制剂TSA介导Ku70乙酰化对结肠癌HT29细胞凋亡和自噬的影响

目的:探讨不同浓度组蛋白去乙酰化酶抑制剂TSA对结肠癌HT29细胞的增殖、凋亡和自噬影响及其机制研究。方法:取对数生长期人结肠癌HT29细胞,采用MTT法检测不同浓度TSA处理对其细胞活力影响,并根据IC50值确定适宜给药浓度;采用流式细胞术检测不同浓度TSA处理后结肠癌HT29细胞的凋亡情况;Western blot验证空白对照组与TSA给药处理组中凋亡标志蛋白Ku70、acetrl-Ku70、Caspase3、Bax、Bcl-2和自噬标志蛋白LC3和Beclin1的表达。结果:MTT法实验结果表明TSA对结肠癌HT29细胞具有时间和浓度依赖性抑制作用,根据IC50=1.12μM,本研究中TSA的给药浓度为0.5μM和1μM;流式细胞凋亡检测结果表明TSA能够显著促进结肠癌HT29细胞凋亡,且其促凋亡作用存在浓度依赖性;此外,Western blot检测结果证实,与空白对照组相比,TSA给药处理可显著上调上述细胞中acetrl-Ku70以及促凋亡蛋白Caspase3、Bax和自噬标志蛋白LC3和Beclin1的表达,下调抗凋亡蛋白Bcl-2的表达(P<0.05)。结论:组蛋白去乙酰化酶抑制剂(TSA)的体外抗结肠癌细胞的增殖、促进细胞凋亡和自噬作用与其上调Ku70蛋白乙酰化密切相关,有望成为临床潜在抗癌靶点。     

Role of calcium in apoptosis of HL-60 cells induced by harringtonine

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Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.     

Midazolam induces cellular apoptosis in human cancer cells and inhibits tumor growth in xenograft mice

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosisinducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-X_L and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.     

Widdrol from <Emphasis Type="Italic">Juniperus chinensis</Emphasis> induces apoptosis in human colon adenocarcinoma HT29 Cells

Juniperus chinensis is a well-known folk remedy in Korea, shown to exhibit anti-tumor, anti-bacterial, anti-fungal, abortifacient, anti-platelet, vasorelaxing, and anti-viral activities. In this study, we report the cytotoxic activity as well as the apoptotic mechanism of widdrol extracted from Juniperus chinensis against the human colon adenocarcinoma cell line HT29. Treatment of HT29 cells with various concentrations of widdrol inhibited growth and induced apoptosis in a dose-dependent manner, as determined by cell viability, chromatin condensation, and sub-G1 phase accumulation. Western blot analysis revealed that apoptosis of HT29 cells was associated with release of cytochrome C from mitochondria into the cytosol, activation of caspases-3, -8, and -9, and proteolytic cleavage of PARP. These results suggest that widdrol exhibits an anti-proliferative effect on HT29 cells via apoptosis and can be a potential candidate in the field of anti-cancer drug discovery.     

基于CRISPR/Cas9技术AEG-1基因敲除神经细胞系的构建及AEG-1基因敲除介导的神经细胞周期阻滞和细胞凋亡抑制

星形胶质细胞上调基因-1(AEG-1)是近年来研究较多的癌基因,但在神经系统疾病方面研究尚少。AEG-1与神经退行性疾病有关,然而其具体作用机制尚不明确。本研究通过设计靶向AEG-1 sgRNA序列并合成相应寡核苷酸,将其克隆到GV392质粒中,构建sgRNA/Cas9二合一表达载体,并进行慢病毒包装纯化。用慢病毒感染小鼠海马神经元HT22细胞,进行药物筛选和sgRNA活性鉴定,建立稳定的AEG-1基因敲除的细胞系;并进一步观察神经元HT22细胞的增殖与凋亡能力。结果显示,成功构建了3种靶向AEG-1基因的sgRNA/Cas9二合一表达载体。所设计的sgRNA的插入序列和开放阅读框架完全正确,成功建立了AEG-1基因敲除的稳转神经细胞系。进一步研究表明,AEG-1敲除后的神经HT22细胞与正常神经HT22细胞相比,细胞突起数目减少, 细胞周期阻滞,细胞凋亡率减少。以上结果为后续进一步研究AEG-1与神经系统疾病关系奠定了基础。     

A let-7/Fas double-negative feedback loop regulates human colon carcinoma cells sensitivity to Fas-related apoptosis

Interferon-γ (IFN-γ) is considered essential for the regulation of anti-tumor reactions as it sensitizes Fas-related apoptosis in HT29 cells, but the mechanism is unclear. In the current study, our data demonstrated that IFN-γ stimulation and Fas activation suppressed Dicer processing and let-7 microRNA biogenesis, while let-7 microRNA strongly inhibited Fas expression by directly targeting Fas mRNA. Accordingly, our results indicate that Fas and let-7 microRNAs form a double-negative feedback loop in IFN-γ and Fas induced apoptosis in colon carcinoma cell line HT29, which may be an important synergistic mechanism in anti-tumor immune response. We also found that a let-7 microRNA inhibitor increased Fas expression and sensitized cells to Fas-related apoptosis, which may have future implications in colon carcinoma therapy.     

Stimulation of GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithelial cells: activation of Nrf2 and JNK-p62/SQSTM1 pathways

The Nrf2-Keap1 pathway is believed to be a critical regulator of the phase II defense system against oxidative stress. By activation of Nrf2, cytoprotective genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase (NQO-1) and γ-glutamyl-cysteine ligase (GCL) are induced. GCL-induced glutathione (GSH) production is believed to affect redox signaling, cell proliferation and death. We here report that tert-butyl hydroperoxide (t-BHP)-induced GSH reduction led to mitochondrial membrane potential loss and apoptosis in cultured human retinal pigment epithelial cells from the ARPE-19 cell line. Hydroxytyrosol (HT), a natural phytochemical from olive leaves and oil, was found to induce phase II enzymes and GSH, thus protect t-BHP-induced mitochondrial dysfunction and apoptosis. Depletion of GSH by buthionine-[S,R]-sulfoximine enhanced t-BHP toxicity and abolished HT protection. Overexpression of Nrf2 increased GSH content and efficiently protected t-BHP-induced mitochondrial membrane potential loss. Meanwhile, HT-induced GSH enhancement and induction of Nrf2 target gene (GCLc, GCLm, HO-1, NQO-1) messenger RNA (mRNA) were inhibited by Nrf2 knockdown, suggesting that HT increases GSH through Nrf2 activation. In addition, we found that HT was able to activate the PI3/Akt and mTOR/p70S6-kinase pathways, both of which contribute to survival signaling in stressed cells. However, the effect of HT was not inhibited by the PI3K inhibitor LY294002. Rather, c-Jun N-terminal kinase (JNK) activation was found to induce p62/SQSTM1 expression, which is involved in Nrf2 activation. Our study demonstrates that Nrf2 activation induced by the JNK pathway plays an essential role in the mechanism behind HT's strengthening of the antiapoptotic actions of the endogenous antioxidant system.     

Hyperthermia induced NFκB mediated apoptosis in normal human monocytes

Conceptual approaches of heat-induced cytotoxic effects against tumor cells must address factors affecting therapeutic index, i.e., the relative toxicity for neoplastic versus normal tissues. Accordingly, we investigated the effect of hyperthermia treatment (HT) on the induction of DNA fragmentation, apoptosis, cell-cycle distribution, NFκB mRNA expression, DNA-binding activity, and phosphorylation of IκBα in the normal human Mono Mac 6 (MM6) cells. For HT, cells were exposed to 43°C. FACS analysis showed a 48.5% increase in apoptosis, increased S-phase fraction, and reduced G2 phase fraction after 43°C treatments. EMSA analysis showed a dose-dependent inhibition of NFκB DNA-binding activity after HT. This HT-mediated inhibition of NFκB was persistent even after 48 h. Immunoblotting analysis revealed dose-dependent inhibition of IκBα phosphorylation. Similarly, RPA analysis showed that HT persistently inhibits NFκB mRNA. These results demonstrate that apoptosis upon HT exposure of MM6 cells is regulated by IκBα phosphorylation mediated suppression of NFκB.     

Rottlerin induces autophagy and apoptotic cell death through a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells: the protective role of autophagy in apoptosis

Rottlerin is widely used as a protein kinase C-delta inhibitor. Recently, several reports have shown the possible apoptosis-inducing effect of rottlerin in some cancer cell lines. Here we report that rottlerin induces not only apoptosis but also autophagy via a PKC-delta-independent pathway in HT1080 human fibrosarcoma cells. Rottlerin treatment induced a dose- and time-dependent inhibition of cell growth, and cytoplasmic vacuolations were markedly shown. These vacuoles were identified as acidic autolysosomes by electron microscopy, acidic vesicular organelle (AVO) staining and transfection of green fluorescent protein-LC3. The LC3-II protein level also increased after treatment with rottlerin. Prolonged exposure to rottlerin eventually caused apoptosis via loss of mitochondrial membrane potential and translocation of AIF from mitochondria to the nucleus. However, the activities of caspase-3, -8 and -9 were not changed, and PARP did not show signs of cleavage. Interestingly, the pretreatment of cells with a specific inhibitor of autophagy (3-methyladenine) accelerated rottlerin-induced apoptosis as revealed by an analysis of the subdiploid fraction and TUNEL assay. Nevertheless, the knockdown of PKC-delta by RNA interference neither affected cell growth nor acidic vacuole formation. Similarly, rottlerin-induced cell death was not prevented by PKC-delta overexpression. Taken together, these findings suggest that rottlerin induces early autophagy and late apoptosis in a PKC-delta-independent manner, and the rottlerin-induced early autophagy may act as a survival mechanism against late apoptosis in HT1080 human fibrosarcoma cells.     

Cox-2 is needed but not sufficient for apoptosis induced by Cox-2 selective inhibitors in colon cancer cells

The role of Cox-2 in NSAID-induced apoptosis is debated. We studied the role of Cox-2 inhibition in apoptosis induced by a selective Cox-2 inhibitor, SC236 (a structural analogue of celecoxib) in two colon cancer cell lines, HT29 (expressing Cox-2 protein) and HCT116 (not expressing Cox-2 protein). Apoptosis was quantified by flow cytometry. SC236 0–75 M decreased cell numbers and induced apoptosis to identical levels in HT29 and HCT116 cells. However, SC236, concentrations >75 M reduced Cox-2 protein expression in HT29 cells and induced greater levels of apoptosis in HT29 than in HCT116 cells. In contrast, sulindac sulfide (SSD) (which inhibits Cox-1 and Cox-2) 0–200 M or sulindac sulfone (SSN) 0–500 M (without significant activity against Cox-1 or Cox-2) caused identical decreases in cell number and increases in apoptosis in HT29 and HCT116 cells. Neither SSD nor SSN altered the expression of Cox-2 in HT29 cells. To determine that the higher levels of apoptosis in HT29 cells with SC236 >75 M were related to decreased Cox-2 protein levels, we decreased Cox-2 protein expression in HT29 cells with curcumin (diferuloylmethane) and studied its effect on SC236-induced apoptosis. Curcumin augmented apoptosis induced by SC236 in HT29 cells but not in Cox-2 lacking HCT116 cells. In conclusion, selective Cox-2 inhibitors can induce apoptosis independent of Cox-2 expression. However they may selectively target cells that express Cox-2 by decreasing their Cox-2 protein expression.     

(2Alpha,3beta)-2,3-dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells

Triterpenoids are known to induce apoptosis and to be anti-tumoural. Maslinic acid, a pentacyclic triterpene, is present in high concentrations in olive pomace. This study examines the response of HT29 and Caco-2 colon-cancer cell lines to maslinic-acid treatment. At concentrations inhibiting cell growth by 50-80% (IC50HT29=61+/-1 microM, IC80HT29=76+/-1 microM and IC50Caco-2=85+/-5 microM, IC80Caco-2=116+/-5 microM), maslinic acid induced strong G0/G1 cell-cycle arrest and DNA fragmentation, and increased caspase-3 activity. However, maslinic acid did not alter the cell cycle or induce apoptosis in the non-tumoural intestine cell lines IEC-6 and IEC-18. Moreover, maslinic acid induced cell differentiation in colon adenocarcinoma cells. These findings support a role for maslinic acid as a tumour suppressant and as a possible new therapeutic tool for aberrant cell proliferation in the colon. In this report, we demonstrate for the first time that, in tumoural cancer cells, maslinic acid exerts a significant anti-proliferation effect by inducing an apoptotic process characterized by caspase-3 activation by a p53-independent mechanism, which occurs via mitochondrial disturbances and cytochrome c release.     

Nitric oxide prevents anoxia-induced apoptosis in colonic HT29 cells.

Apoptosis is a critical determinant of tissue mass homeostasis and may play a role in carcinogenesis. The aim of the present study was to investigate anoxia-induced cell death in colon-derived HT29 cells and the effect of nitric oxide on this phenomenon. It was found that HT29 cells subjected to anoxia undergo apoptosis in a time dependent manner, as determined by DNA fragmentation and Hoechst-33258 dye staining. Cytochrome c release from mitochondria to cytosol is a key step in this process and this release precedes DNA fragmentation. NO inhibits anoxia induced apoptosis in these cells by inhibiting the release of cytochrome c and thus may play a role in modulating the apoptotic cell death of colon-derived epithelial cells.     

SOX-17 is involved in invasion and apoptosis of colorectal cancer cells through regulating miR-302b-3p expression

The prognosis of advanced colorectal cancer (CRC) is currently still very poor, which suggests that the biological mechanisms of CRC oncogenesis are not fully understood. This study was conducted to explore the regulatory effect of SOX-17 on the expression of microRNA (miR)-302b-3p, and the involvement of SOX-17 in the invasion and apoptosis of CRC cells. The expression of SOX-17 and miR-302a,b,c,d-3p in colorectal cancer and normal colon epithelial cell lines was measured by real-time polymerase chain reaction and/or western blot. The regulatory effects of SOX-17 on miR-302b-3p gene in HT29 and LoVo cells were tested using the ChiP assay. The biological activities of SOX-17 and miR-302b-3p were evaluated by invasion and apoptosis assay. Results showed that transfection of SOX-17 small interfering RNA (siSOX-17) significantly increased, whereas transfection of SOX-17 overexpression vector (oeSOX-17) significantly decreased, miR-302b expression in HT29 and LoVo cells. Cotransfection of oeSOX-17 and miR-302b-3p inhibitor (INmiR-302b) significantly blocked the effects of SOX-17 in HT29 and LoVo cells. ChIP experiments showed that SOX-17 bonded to the miR-302b-3p promoter in HT29 and LoVo cells. Transfection of oeSOX-17 and miR-302b-3p mimics (MImiR-302b) significantly decreased, whereas transfection of siSOX-17 and INmiR-302b significantly increased, the invasion of HT29 and LoVo cells. In contrast, transfection of oeSOX-17 and MImiR-302b significantly increased, while transfection of siSOX-17 and INmiR-302b significantly decreased, apoptosis in HT29 and LoVo cells. Cotransfection of oeSOX-17 and INmiR-302b significantly blocked the effects of oeSOX-17 on cell invasion and apoptosis in HT29 and LoVo cells. These results suggested that SOX-17 can bind to the promoter of miR-302b-3p gene to regulate its expression, while both SOX-17 and miR-302b regulate the invasion and apoptosis in colorectal cancer cells.     

Isolation and partial characterisation of a novel lectin from Aegle marmelos fruit and its effect on adherence and invasion of Shigellae to HT29 cells

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 μg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.     

RasV12 induces Survivin/AuroraB pathway conferring tumor cell apoptosis resistance

Several cancers are treated by interferons α and γ in association with conventional chemotherapy due to the resistance observed with interferon treatment alone. The frequency of un-sensitive cancer depends on tumor origin and oncogenic genes. Preclinical studies have highlighted interferon resistance in many cancers such as colon carcinoma due to oncogenic Ras. However, the resistance mechanism remains elusive. Apoptosis and proliferation of Ras^wt and mutated Ras^V12 transformed colon carcinoma cells treated with several recombinant interferon combinations were analyzed by flow cytometer and immunoblot. Apoptotic pathways of resistant Ras^V12 cells were investigated using siRNA strategy to determine key proteins involved in this process. We show that interferons α and γ synergized to induce human Ras^wt colon carcinoma cell (HT29) apoptosis by caspases and PARP-1 cleavages in contrast to Ras^V12 mutated colon carcinoma cells (SW480, HT29 clone). However, Ras^V12 siRNA restored interferon sensitivity of Ras^V12-HT29 clone to apoptosis. Survivin siRNA increased interferon apoptosis in Ras^wt cells demonstrating the key role of this protein in cell survival. Ras^V12 mutation in HT29 clone neutralized the interferon effect on Survivin suppression and maintained high level of phospho-Aurora-B/Histone H3, which protected cells from apoptosis. SiRNA strategy against both Aurora-B and Survivin in Ras^V12 cells synergized to restore interferon -induced apoptosis. Ras^V12 cells are less sensitive than Ras^wt cells to interferon induced cell apoptosis due to a Survivin/Aurora-B survival alternative pathway. Taken together, these results may provide interest in siRNA-therapeutic strategy and diagnostic relevance for therapy.     

Interferon-gamma sensitizes colonic epithelial cell lines to physiological and therapeutic inducers of colonocyte apoptosis

Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell's sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis.     

Quinone-induced apoptosis in human colon adenocarcinoma cells via DT-diaphorase mediated bioactivation

DT-diaphorase (DTD) activity has been related to bioactivation and cytotoxicity of antitumor quinones. A pair of human colon adenocarcinoma cell lines, HT29 and BE, were used in this study to examine the role of DTD in antitumor quinone induced apoptosis. HT29 cells have elevated levels of DTD whereas BE cells lack functional DTD due to a point mutation which results in a complete lack of DTD activity. MeDZQ, a quinone that is efficiently bioactivated by DTD, induced apoptosis both in HT29 and BE cells, but with a much higher incidence in HT29, as assessed by morphological criteria and the formation of oligonucleosomal fragments of DNA. Two other quinone compounds which are also substrates for DTD, i.e. streptonigrin and mitomycin C, also preferentially induced apoptosis in HT29 cells, which could be inhibited by dicoumarol. Our data suggest that bioreductive activation of antitumor quinones by DTD results in induction of apoptosis in human colon carcinoma cells.