TRAIL-Mediated Apoptosis in Breast Cancer Cells Cultured as 3D Spheroids

TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44^hiCD24^loALDH1^hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE₂) pathway. We show that inhibition of the COX-2/PGE₂ pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44^hiCD24^lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.     

诱杀受体3——肿瘤基因治疗的新靶点

诱杀受体3（DcR3）又称肿瘤坏死因子受体（TNFR）6B,是最近发现的TNFR超家族成员之一。DcR3是一种凋亡抑制蛋白,能够抑制肿瘤细胞凋亡,促进肿瘤细胞免疫逃避。研究显示,DcR3在人类正常分化组织中很少表达,却特异性表达在常见的肿瘤,它的表达上调可预示肿瘤恶性程度和预后。作为肿瘤分子标志物,体液及组织中高水平的DcR3表达能够辅助肿瘤诊断和治疗监测。应用分子生物技术可以抑制肿瘤细胞中DcR3的表达,从而诱导肿瘤细胞的凋亡。     

Decoy Receptor 3 Improves Survival in Experimental Sepsis by Suppressing the Inflammatory Response and Lymphocyte Apoptosis

Purpose

Unbalanced inflammatory response and lymphocyte apoptosis is associated with high mortality in septic patients. Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, is an anti-inflammatory and anti-apoptotic factor. Recently, DcR3 expression was found to be increased in septic patients. This study evaluated the therapeutic effect and mechanisms of DcR3 on cecal ligation and puncture (CLP)-induced sepsis in mice.

Methods

C57BL/6 mice were subjected to CLP-induced polymicrobial sepsis. DcR3 Fc was intravenously injected 30 min before and 6 h after CLP. Bacterial clearance, cytokine production, histology, lymphocyte apoptosis and survival were evaluated. Furthermore, we investigated the systemic effects of DcR3 in in vitro lymphocyte apoptosis regulation.

Results

Our results demonstrated that DcR3 protein treatments significantly improved survival in septic mice (p <0.05). Treatment with DcR3 protein significantly reduced the inflammatory response and decreased lymphocyte apoptosis in the thymus and spleen. Histopathological findings of the lung and liver showed milder impairment after DcR3 administration. In vitro experiments showed that DcR3 Fc inhibited Fas-FasL mediated lymphocyte apoptosis.

Conclusions

Treatment with the DcR3 protein protects mice from sepsis by suppressing the inflammatory response and lymphocyte apoptosis. DcR3 protein may be useful in treatment of sepsis.     

Cathepsin Inhibition-Induced Lysosomal Dysfunction Enhances Pancreatic Beta-Cell Apoptosis in High Glucose

Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.     

雷公藤甲素诱导胰腺癌细胞凋亡

目的：观察雷公藤甲素对胰腺癌细胞BxPC-3和PANC-1生长抑制及诱导细胞凋亡作用,探讨雷公藤甲素抗胰腺癌的机制。方法：雷公藤甲素处理BxPC-3和PANC—1细胞后,用M1rr法检测细胞的生长抑制,用流式细胞术检测细胞的凋亡率,用罗丹明123和DCFH—DA染色方法测定细胞线粒体膜跨膜电位变化和活性氧（ROS）的产生,用Western印迹检测Bcl-2、Bax蛋白表达的变化。结果：雷公藤甲素对胰腺癌细胞BxPC-3和PANC—1具有生长抑制和诱导细胞凋亡的作用,且呈时间和剂量依赖关系;处理72h后,胰腺癌细胞线粒体跨膜电位明显下降,Bax表达上调,Bcl-2表达下降。结论：雷公藤甲素能有效抑制胰腺癌细胞增殖,通过增强线粒体通透性诱导细胞凋亡。     

西达本胺诱导胰腺癌细胞凋亡

目的：观察西达本胺对胰腺癌细胞BxPC-3和PANC-1生长抑制及诱导细胞凋亡作用,探讨西达本胺抗胰腺癌的机制。方法：西达本胺处理BxPC-3和PANC-1细胞后,用流式细胞术检测细胞的凋亡率,用罗丹明123和DCFH—DA染色方法测定细胞线粒体膜跨膜电位变化和活性氧（ROS）的产生,用Western印迹检测Bcl-2家族和γH2AX蛋白表达的变化。结果：西达本胺对胰腺癌细胞BxPC-3和PANC-1具有生长抑制和诱导细胞凋亡的作用,且呈时间和剂量依赖关系;处理72h后,胰腺癌细胞内ROS产生增强导致DNA损伤发生,且线粒体跨膜电位明显下降;促凋亡蛋白Bax的表达,抑制抑凋亡蛋白Bcl-2和Mcl—1的表达。结论：西达本胺具有抑制胰腺癌细胞增殖,诱导细胞凋亡的作用;西达本胺增强胰腺癌细胞内ROS的产生并导致DNA损伤,最终诱导细胞凋亡的发生。     

Resveratrol Enhances Palmitate-Induced ER Stress and Apoptosis in Cancer Cells

Background

Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several in vitro and in vivo studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines.

Principal Findings

We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity.

Conclusions

Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by ER stress through a CHOP-mediated apoptotic process and may represent a potential anticancer strategy.     

ATP-Citrate Lyase Reduction Mediates Palmitate-induced Apoptosis in Pancreatic Beta Cells

Elevated extracellular lipids, such as the free fatty acid palmitate, can induce pancreatic beta cell endoplasmic reticulum (ER) stress and apoptosis, thereby contributing to the initiation and progression of type 2 diabetes. ATP-citrate lyase (ACLY), a key enzyme in cellular lipid production, was identified as a palmitate target in a proteomic screen. We investigated the effects of palmitate on ACLY activity and phosphorylation and its role in beta cell ER stress and apoptosis. We demonstrated that treatment of MIN6 cells, mouse islets and human islets with palmitate reduced ACLY protein levels. These in vitro results were validated by our finding that islets from high fat-fed mice had a significant decrease in ACLY, similar to that previously observed in type 2 diabetic human islets. Palmitate decreased intracellular acetyl-CoA levels to a similar degree as the ACLY inhibitor, SB-204990, suggesting a reduction in ACLY activity. ACLY inhibitors alone were sufficient to induce CCAAT/enhancer-binding protein homologues protein (CHOP)-dependent ER stress and caspase-3-dependent apoptosis. Similarly, even modest shRNA-mediated knockdown of ACLY caused a significant increase in beta cell apoptosis and ER stress. The effects of chemical ACLY inhibition and palmitate were non-additive and therefore potentially mediated by a common mechanism. Indeed, overexpression of ACLY prevented palmitate-induced beta cell death. These observations provide new evidence that ACLY expression and activity can be suppressed by exogenous lipids and demonstrate a critical role for ACLY in pancreatic beta cell survival. These findings add to the emerging body of evidence linking beta cell metabolism with programmed cell death.     

Bmi1 Enhances Tumorigenicity and Cancer Stem Cell Function in Pancreatic Adenocarcinoma

Background

Bmi1 is an integral component of the Polycomb Repressive Complex 1 (PRC1) and is involved in the pathogenesis of multiple cancers. It also plays a key role in the functioning of endogenous stem cells and cancer stem cells. Previous work implicated a role for cancer stem cells in the pathogenesis of pancreatic cancer. We hypothesized that Bmi1 plays an integral role in enhancing pancreatic tumorigenicity and the function of cancer stem cells in pancreatic ductal adenocarcinoma.

Methods

We measured endogenous Bmi1 levels in primary human pancreatic ductal adenocarcinomas, pancreatic intraepithelial neoplasias (PanINs) and normal pancreas by immunohistochemistry and Western blotting. The function of Bmi1 in pancreatic cancer was assessed by alteration of Bmi1 expression in several cell model systems by measuring cell proliferation, cell apoptosis, in vitro invasion, chemotherapy resistance, and in vivo growth and metastasis in an orthotopic model of pancreatic cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human pancreatic cancer xenografts after Bmi1 silencing.

Results

Bmi1 was overexpressed in human PanINs, pancreatic cancers, and in several pancreatic cancer cell lines. Overexpression of Bmi1 in MiaPaCa2 cells resulted in increased proliferation, in vitro invasion, larger in vivo tumors, more metastases, and gemcitabine resistance while opposite results were seen when Bmi1 was silenced in Panc-1 cells. Bmi1 was overexpressed in the cancer stem cell compartment of primary human pancreatic cancer xenografts. Pancreatic tumorspheres also demonstrated high levels of Bmi1. Silencing of Bmi1 inhibited secondary and tertiary tumorsphere formation, decreased primary pancreatic xenograft growth, and lowered the proportion of cancer stem cells in the xenograft tissue.

Conclusions

Our results implicate Bmi1 in the invasiveness and growth of pancreatic cancer and demonstrate its key role in the regulation of pancreatic cancer stem cells.     

Inhibition of Amyloid Precursor Protein Processing Enhances Gemcitabine-mediated Cytotoxicity in Pancreatic Cancer Cells

Pancreatic adenocarcinoma or pancreatic cancer is often diagnosed at a very late stage at which point treatment options are minimal. Current chemotherapeutic interventions prolong survival marginally, thereby emphasizing the acute need for better treatment options to effectively manage this disease. Studies from different laboratories have shown that the Alzheimer disease-associated amyloid precursor protein (APP) is overexpressed in various cancers but its significance is not known. Here we sought to determine the role of APP in pancreatic cancer cell survival and proliferation. Our results show that pancreatic cancer cells secrete high levels of sAPPα, the α-secretase cleaved ectodomain fragment of APP, as compared with normal non-cancerous cells. Treatment of cells with batimastat or GI254023X, inhibitors of the α-secretase ADAM10, prevented sAPPα generation and reduced cell survival. Additionally, inhibition of sAPPα significantly reduced anchorage independent growth of the cancer cells. The effect of batimastat on cell survival and colony formation was enhanced when sAPPα downregulation was combined with gemcitabine treatment. Moreover, treatment of batimastat-treated cells with recombinant sAPPα reversed the inhibitory effect of the drug thereby indicating that sAPPα can indeed induce proliferation of cancer cells. Down-regulation of APP and ADAM10 brought about similar results, as did batimastat treatment, thereby confirming that APP processing is important for growth and proliferation of these cells. These results suggest that inhibition of sAPPα generation might enhance the effectiveness of the existing chemotherapeutic regimen for a better outcome.     

miR-451a促进胰腺癌细胞对吉西他滨的敏感性

目的:探究miR-451a在胰腺癌吉西他滨耐药中的功能。方法:通过低浓度梯度递增法建立胰腺癌吉西他滨耐药细胞株,microRNA(miRNA)测序筛选耐药相关miRNA;细胞存活曲线、克隆形成实验及流式凋亡实验分析miR-451a对胰腺癌细胞耐药的影响;裸鼠成瘤实验检测在动物体内模型中miR-451a对胰腺癌吉西他滨耐药的调控作用;调取TCGA数据分析miR-451a表达水平与胰腺癌病人预后的相关性。结果:胰腺癌吉西他滨耐药细胞株中miR-451a表达水平明显下调;miR-451a过表达增加胰腺癌细胞对吉西他滨的敏感性,增强了吉西他滨抑制细胞增殖和诱导细胞凋亡的作用;miR-451a诱导了裸鼠皮下肿瘤对吉西他滨敏感;miR-451a低表达与胰腺癌患者不良预后相关。结论:miR-451a增强了胰腺癌细胞对吉西他滨的敏感性。     

PI3K Inhibition Enhances Doxorubicin-Induced Apoptosis in Sarcoma Cells

We searched for a drug capable of sensitization of sarcoma cells to doxorubicin (DOX). We report that the dual PI3K/mTOR inhibitor PI103 enhances the efficacy of DOX in several sarcoma cell lines and interacts with DOX in the induction of apoptosis. PI103 decreased the expression of MDR1 and MRP1, which resulted in DOX accumulation. However, the enhancement of DOX-induced apoptosis was unrelated to DOX accumulation. Neither did it involve inhibition of mTOR. Instead, the combination treatment of DOX plus PI103 activated Bax, the mitochondrial apoptosis pathway, and caspase 3. Caspase 3 activation was also observed in xenografts of sarcoma cells in nude mice upon combination of DOX with the specific PI3K inhibitor GDC-0941. Although the increase in apoptosis did not further impact on tumor growth when compared to the efficient growth inhibition by GDC-0941 alone, these findings suggest that inhibition of PI3K may improve DOX-induced proapoptotic effects in sarcoma. Taken together with similar recent studies of neuroblastoma- and glioblastoma-derived cells, PI3K inhibition seems to be a more general option to sensitize tumor cells to anthracyclines.     

Examination of Apoptosis Signaling in Pancreatic Cancer by Computational Signal Transduction Analysis

Background

Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of cancer death. Changes in apoptosis signaling in pancreatic cancer result in chemotherapy resistance and aggressive growth and metastasizing. The aim of this study was to characterize the apoptosis pathway in pancreatic cancer computationally by evaluation of experimental data from high-throughput technologies and public data bases. Therefore, gene expression analysis of microdissected pancreatic tumor tissue was implemented in a model of the apoptosis pathway obtained by computational protein interaction prediction.

Methodology/Principal Findings

Apoptosis pathway related genes were assembled from electronic databases. To assess expression of these genes we constructed a virtual subarray from a whole genome analysis from microdissected native tumor tissue. To obtain a model of the apoptosis pathway, interactions of members of the apoptosis pathway were analysed using public databases and computational prediction of protein interactions. Gene expression data were implemented in the apoptosis pathway model. 19 genes were found differentially expressed and 12 genes had an already known pathophysiological role in PDAC, such as Survivin/BIRC5, BNIP3 and TNF-R1. Furthermore we validated differential expression of IL1R2 and Livin/BIRC7 by RT-PCR and immunohistochemistry. Implementation of the gene expression data in the apoptosis pathway map suggested two higher level defects of the pathway at the level of cell death receptors and within the intrinsic signaling cascade consistent with references on apoptosis in PDAC. Protein interaction prediction further showed possible new interactions between the single pathway members, which demonstrate the complexity of the apoptosis pathway.

Conclusions/Significance

Our data shows that by computational evaluation of public accessible data an acceptable virtual image of the apoptosis pathway might be given. By this approach we could identify two higher level defects of the apoptosis pathway in PDAC. We could further for the first time identify IL1R2 as possible candidate gene in PDAC.     

Sensitization of Human Pancreatic Cancer Cells Harboring Mutated K-ras to Apoptosis

Pancreatic cancer is a devastating human malignancy and gain of functional mutations in K-ras oncogene is observed in 75%-90% of the patients. Studies have shown that oncogenic ras is not only able to promote cell growth or survival, but also apoptosis, depending upon circumstances. Using pancreatic cancer cell lines with or without expressing mutated K-ras, we demonstrated that the inhibition of endogenous PKC activity sensitized human pancreatic cancer cells (MIA and PANC-1) expressing mutated K-ras to apoptosis, which had no apoptotic effect on BxPC-3 pancreatic cancer cells that contain a normal Ras as well as human lung epithelial BAES-2B cells. In this apoptotic process, the level of ROS was increased and PUMA was upregulated in a p73-dependent fashion in MIA and PANC-1 cells. Subsequently, caspase-3 was cleaved. A full induction of apoptosis required the activation of both ROS- and p73-mediated pathways. The data suggest that PKC is a crucial factor that copes with aberrant K-ras to maintain the homeostasis of the pancreatic cancer cells harboring mutated K-ras. However, the suppression or loss of PKC disrupts the balance and initiates an apoptotic crisis, in which ROS and p73 appear the potential, key targets.     

Irinotecan Upregulates Fibroblast Growth Factor Receptor 3 Expression in Colorectal Cancer Cells,Which Mitigates Irinotecan-Induced Apoptosis

BACKGROUND: Irinotecan (IRI) is an integral part of colorectal cancer (CRC) therapy, but response rates are unsatisfactory and resistance mechanisms are still insufficiently understood. As fibroblast growth factor receptor 3 (FGFR3) mediates essential survival signals in CRC, it is a candidate gene for causing intrinsic resistance to IRI. METHODS: We have used cell line models overexpressing FGFR3 to study the receptor's impact on IRI response. For pathway blockade, a dominant-negative receptor mutant and a small molecule kinase inhibitor were employed. RESULTS: IRI exposure induced expression of FGFR3 as well as its ligands FGF8 and FGF18 both in cell cultures and in xenograft tumors. As overexpression of FGFR3 mitigated IRI-induced apoptosis in CRC cell models, this suggests that the drug itself activated a survival response. On the cellular level, the antiapoptotic protein bcl-xl was upregulated and caspase 3 activation was inhibited. Targeting FGFR3 signaling using a dominant-negative receptor mutant sensitized cells for IRI. In addition, the FGFR inhibitor PD173074 acted synergistically with the chemotherapeutic drug and significantly enhanced IRI-induced caspase 3 activity in vitro. In vivo, PD173074 strongly inhibited growth of IRI-treated tumors. CONCLUSION: Together, our results indicate that targeting FGFR3 can be a promising strategy to enhance IRI response in CRC patients.