Sensitization of pancreatic carcinoma cells for gamma-irradiation-induced apoptosis by XIAP inhibition

Resistance of pancreatic cancer to current treatments including radiotherapy remains a major challenge in oncology and may be caused by defects in apoptosis programs. Since 'inhibitor of apoptosis proteins' (IAPs) block apoptosis at the core of the apoptotic machinery by inhibiting caspases, therapeutic modulation of IAPs could tackle a key resistance mechanism. Here, we report that targeting X-linked inhibitor of apoptosis (XIAP) by RNA-interference-mediated knockdown or overexpression of second mitochondria-derived activator of caspase significantly enhanced apoptosis and markedly reduced clonogenic growth of pancreatic carcinoma cells upon gamma-irradiation. Analysis of signaling pathways revealed that antagonizing XIAP increased activation of caspase-2, -3, -8 and -9 and loss of mitochondrial membrane potential upon gamma-irradiation. Interestingly, inhibition of caspases also reduced the cooperative effect of XIAP targeting and gamma-irradiation to trigger mitochondrial perturbations, suggesting that XIAP controls a feedback mitochondrial amplification loop by regulating caspase activity. Importantly, our data demonstrate for the first time that small molecule XIAP inhibitors sensitized pancreatic carcinoma cells for gamma-irradiation-induced apoptosis, whereas they had no effect on gamma-irradiation-mediated apoptosis of non-malignant fibroblasts indicating some tumor specificity. In conclusion, targeting XIAP, for example by small molecules, is a promising novel approach to enhance radiosensitivity of pancreatic cancer that warrants further investigation.     

HDAC2 attenuates TRAIL-induced apoptosis of pancreatic cancer cells

Background  

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors with a dismal prognosis and no effective conservative therapeutic strategies. Although it is demonstrated that histone deacetylases (HDACs), especially the class I HDACs HDAC1, 2 and 3 are highly expressed in this disease, little is known about HDAC isoenzyme specific functions.     

蟾毒灵通过下调c-Flip和XIAP增强TRAIL诱导的乳腺癌MDA-MB-231细胞凋亡

目的：研究蟾毒灵（ bufalin）对TRAIL诱导乳腺癌MDA-MB-231细胞凋亡的的影响及机制。方法：MTT法测定细胞活力、采用流式细胞仪检测细胞凋亡,免疫荧光实验检测DR4、DR5的表达情况,Western blot法检测c-Flip和XIAP蛋白的表达。结果：MTT结果显示bufalin作用于MDA-MB-231细胞后24h和48h的IC50分别为934nmol/L 和513nmol/L。流式分析显示50nmol/L 的 bufalin 作用24h 仅诱导4.5%的MDA-MB-231细胞凋亡,100ng/ml的TRAIL作用24h诱导5.1%的MDA-MB-231细胞凋亡,50nmol/L的bufalin与100ng/ml的TRAIL联合作用24小时后可诱导32.5%的MDA-MB-231细胞凋亡。Western blot分析显示bufalin与TRAIL联合作用后导致c-Flip和XIAP的明显下调。结论：Bufalin可通过下调c-FLIP和XIAP的表达增强TRAIL诱导的乳腺癌MDA-MB-231细胞凋亡。     

SKP2 confers resistance of pancreatic cancer cells towards TRAIL-induced apoptosis

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dismal prognosis and no effective conservative therapy exists. Although the F-box protein S-phase kinase associated protein 2 (SKP2) is highly expressed and regulates cell cycle progression in PDAC, alternative SKP2 functions in PDAC are unknown. Using RNA interference we now demonstrate that SKP2 confers resistance of a subset of PDAC cell lines towards the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), but not the topoisomerase II inhibitor etoposide. We observed accelerated cleavage of the BH3-only protein Bid and augmented downregulation of cFLIPL, XIAP and MCL1 upon treatment of SKP2-depleted MiaPaCa2 cells with TRAIL. Our data disclose a novel SKP2 function in PDAC cells and therefore define SKP2 as a molecular target.     

IFNgamma sensitization to TRAIL-induced apoptosis in human thyroid carcinoma cells by upregulating Bak expression

TRAIL preferentially induces apoptosis in tumor cells and virus-infected cells. Unlike other tumor necrosis factor family members, TRAIL does not kill cells from most normal tissues and has thus been proposed as a promising new cancer treatment. Our study demonstrated that IFNgamma combined with TRAIL can trigger apoptosis in vitro in several resistant thyroid tumor cell lines, such as thyroid anaplastic carcinoma cells (ARO cells), while either agent alone exerts only a minimal effect. We further tested this effect on a mouse thyroid tumor model, when in vivo tumor growth was also significantly inhibited by this combination. The mechanism of how IFNgamma sensitized thyroid carcinoma cells to TRAIL-induced apoptosis was investigated by screening global gene alterations in ARO cells treated with IFNgamma. Microarray data revealed that a proapoptotic gene, Bak, is markedly upregulated by IFNgamma, and this was confirmed by RNase protection assay. Western blot analysis also showed a significant increase in Bak at the protein level. Upregulation of Bak and sensitization for apoptosis by IFNgamma was blocked by overexpression of antisense Bak in ARO cells. Furthermore, overexpression of Bak sensitized ARO cell to TRAIL-induced apoptosis without the need for IFNgamma pretreatment. This suggests that Bak is a regulatory molecule involved in IFNgamma-facilitated TRAIL-mediated apoptosis in thyroid cancer cells.     

Adenovirus-mediated IKKbetaKA expression sensitizes prostate carcinoma cells to TRAIL-induced apoptosis

Despite the fact that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells, TRAIL resistance in cancer cells has challenged the use of TRAIL as a therapeutic agent. First, prostate carcinoma cell lines (DU145, LNCaP and PC3) were screened for sensitivity to adenovirus delivery of TRAIL (Ad5hTRAIL). As amplified Ikappa B kinase (IKK) activity is responsible for the constitutive nuclear factor-kappaB (NF-kappaB) activation leading to uncontrolled cell growth and metastasis, a dual vector approach using both an adenovirus vector (Ad) expressing the dominant-negative mutant of IKKbeta (AdIKKbetaKA) and Ad5hTRAIL was employed to determine if prostate cancer cells were sensitized to TRAIL in the setting of IKK inhibition. Inhibition of the NF-kappaB pathway through IKK blockade sensitized all three prostate cancer cell lines to TRAIL, regardless of NF-kappaB activation or decoy receptor gene expression. Moreover, a novel quantitative real-time RT-PCR assay and conventional flow cytometry analysis indicated that TRAIL-resistant DU145 and LNCaP cells, but not TRAIL-sensitive PC3 cells, expressed substantial amounts of TRAIL Decoy Receptor 4. In conclusion, TRAIL decoy receptor expression appeared to be the chief determinant of TRAIL resistance encountered in prostate carcinoma cell lines.     

Rottlerin sensitizes glioma cells to TRAIL-induced apoptosis by inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP

In the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells, treatment with TRAIL in combination with subtoxic doses of rottlerin induced rapid apoptosis. While the proteolytic processing of procaspase-3 by TRAIL was partially blocked in these cells, treatment with rottlerin efficiently recovered TRAIL-induced activation of caspases. Treatment with rottlerin significantly decreased Cdc2 activity through the downregulation of cyclin A, cyclin B, and Cdc2 proteins, whereas the sensitizing effect of rottlerin on TRAIL-induced apoptosis was independent of PKCdelta activity. Furthermore, treatment with rottlerin downregulated the protein levels of survivin and X-chromosome-linked IAP (XIAP), two major caspase inhibitors. Forced expression of Cdc2 together with cyclin B attenuated rottlerin-potentiated TRAIL-induced apoptosis by over-riding the rottlerin-mediated downregulation of survivin and XIAP protein levels. Taken together, inhibition of Cdc2 activity and the subsequent downregulation of survivin and XIAP by subtoxic doses of rottlerin contribute to amplification of caspase cascades, thereby overcoming resistance of glioma cells to TRAIL-mediated apoptosis. Since rottlerin can sensitize Bcl-2- or Bcl-xL-overexpressing glioma cells but not human astrocytes to TRAIL-induced apoptosis, this combined treatment may offer an attractive strategy for safely treating resistant gliomas.     

胰腺癌组织XIAP蛋白表达预后价值的探讨

目的:探讨胰腺癌组织XIAP蛋白的表达与临床病理特征和生存期的关系。方法:收集胰腺癌组织标本54例及正常胰腺组织14例,并随访其生存期;免疫组织化学检测XIAP的表达;标准评定采用半定量法。结果:54例胰腺癌组织中48例(88.9%)XIAP阳性表达,14例胰腺正常组织中XIAP阳性表达率为50.0%。XIAP表达与胰腺癌组织分化程度呈负相关(P<0.05),而与临床TNM分期无关。XIAP表达与肿瘤是否发生血管浸润和淋巴结转移有关,P<0.05。XIAP表达阴性及弱阳性与表达中等阳性及强阳性比较,术后生存率差异有统计学意义,P=0.041。多因素分析结果显示,TNM分期、XIAP蛋白表达水平为独立预后因子。结论:XIAP表达水平高的胰腺癌组织恶性程度更高,发生血管浸润及淋巴结转移的机会更高;XIAP表达水平与患者术后生存期有关;TNM分期、XIAP蛋白表达水平可作为评价预后的有价值指标。     

A mitochondrial block and expression of XIAP lead to resistance to TRAIL-induced apoptosis during progression to metastasis of a colon carcinoma

A pair of isogenic colon carcinoma cells, SW480 and 620, was used to investigate the mechanisms of acquired tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance during tumour progression. Whereas primary tumour SW480 cells are sensitive to TRAIL-induced apoptosis, metastatic SW620 cells are resistant. The apoptotic signalling activated by TRAIL in SW480 cells is a type II pathway. We show that in SW620 cells, although caspase-8 is recruited and activated at the death-inducing-signalling complex and Bid is cleaved, this does not lead to caspase-9 activation. Comparison of Bcl-2, Bcl-xL and Mcl-1 levels in both cell lines showed no difference. In SW620 cells transfected with a tBid-GFP construct, tBid-GFP was correctly localized to the mitochondria. Thus, the resistance of SW620 cells is at the level of the mitochondria that can withstand large amounts of tBid. Although caspase-3 was directly cleaved by caspase-8 in SW620 cells to yield the p20 fragment, no further autocatalytic maturation into the p17 fragment was observed. We show that, in contrast to SW480 cells, the SW620 cell line expresses high amounts of X-linked inhibitor of apoptosis (XIAP). Downregulation of XIAP with bortezomib or small-interfering RNA was sufficient to restore the sensitivity of SW620 cells to TRAIL-induced apoptosis in the absence of SMAC/Diablo or cytochrome c release from the mitochondria. Thus, SW620 cells have developed a dual resistance to TRAIL-induced apoptosis: a block at the level of the mitochondria and, after a conversion to a type I pathway, an increased expression of XIAP which inhibits this pathway.     

吉西他滨诱导胰腺癌细胞BxPC-3中XIAP基因表达的研究

背景与目的：凋亡抑制蛋白（inhibitor of apoptosis proteins,IAPs）是细胞内一类重要的凋亡抑制因子家族,X染色体连锁的凋亡抑制蛋白（X-linked inhibitor of apoptosis,XIAP）是IAPs家族中重要的一员,在多种肿瘤细胞中高表达,并与肿瘤的化疗耐药相关。本研究旨在探讨吉西他滨（gemcitabine,GEM）诱导胰腺癌细胞BxPC-3中XIAP的表达及其与化疗耐药的关系。方法：3.10μg/mL GEM作用于BxPC-3细胞不同时间后（0、24、36、48和72h）用流式细胞仪分析各实验组细胞周期及凋亡情况（其中0h为对照组）,运用RT-PCR检测XIAP mRNA转录,采用Western blot法检测XIAP蛋白表达。结果：实验组细胞被阻滞于G0/G1期;各实验组细胞凋亡率分别增加至（10.3&#177;1.8）%、（14.2&#177;1.5）%、（18.8&#177;1.7）%和（20.3&#177;2.0）%,实验组与对照组（1.23&#177;1.6）%相比性差异有统计学意义（F=146.24,P〈0.05）,48h组与72h组相比差异无统计学意义（P〉;0.05）。XIAP mRNA转录及蛋白表达均随GEM作用时间延长而增加,实验组较对照组增加,差异有统计学意义（F=83.72,F=103.58,P〈0.05）,24h与36h组间比较,差异无统计学意义（P〉0.05）,36h组与48h组间、48h与72h组间比较,差异均有统计学意义（P〈0.05）。结论：GEM能抑制胰腺癌BxPC-3细胞增殖并促进凋亡,随着作用时间的延长其药物敏感性减低。GEM作用一定时间可诱导XIAP表达增加,从而可能参与并使凋亡受阻,进而促进化疗耐药得以实现。     

Sensitization of chronic lymphocytic leukemia cells to TRAIL-induced apoptosis by hyperthermia

We recently reported that, in cultured leukemic T lymphocytes and promyelocytic cells, a mild heat shock treatment (1 h at 42 degrees C) induced a long lasting stimulation of the apoptosis induced by TNF-related apoptosis inducing ligand (TRAIL). On the opposite, no effects were recorded toward normal human T lymphocytes. The apoptogenic efficiency of TRAIL in leukemic lymphocytes is linked to the long lasting increased ability of TRAIL to recognize and bind DR4 and DR5 receptors during hyperthermia. Here, we have analyzed whether this new apoptotic co-treatment could be relevant toward primary cells from patients suffering of chronic lymphocytic leukemia. Analysis of samples from 24 patients with different ages, sex and disease stages revealed that half of them had lymphocytes that, once isolated and analyzed in vitro, positively responded (increase of cell death) to the heat shock plus TRAIL co-treatment. Analysis of the level of expression of various anti-apoptotic proteins in the cell samples revealed a great heterogeneity between patients and no clear relationships could be drawn. Nevertheless, most cell samples that were sensitive to TRAIL plus heat shock induced apoptosis displayed a higher level of cell surface DR4 and DR5 receptors than the non-sensitive counterparts. Hence, analysis of the level of TRAIL surface receptors is a prerequisite for future clinical applications based on this protocol.     

Chemosensitization of hormone-refractory prostate cancer cells by curcumin to TRAIL-induced apoptosis

Failure to undergo apoptosis has been implicated in the resistance of tumor cells to anticancer therapies. Promotion of apoptosis in tumor cells could potentially increase the efficacy of conventional treatment regimens and improve prognosis. Prostate cancer cells are generally resistant to induction of apoptosis by anticancer agents and death ligands. We investigated the sensitization of prostate cancer cell lines by curcumin (diferuloyl-methane) to TNF-related apoptosis inducing ligand (TRAIL)-induced apoptosis. Prostate cancer cells treated with curcumin or TRAIL or curcumin and TRAIL together were assessed for induction of apoptosis and pathway of apoptosis was determined from the activation of procaspases and release of cytochrome c from mitochondria. Curcumin sensitized LNCaP, DU145 and PC3 tumor cell lines to TRAIL. Combined curcumin and TRAIL treatment produced the most loss of viable cells by inducing apoptosis as revealed by accumulation of hypodiploid cells in sub-G1 phase, enhanced annexin V binding, DNA fragmentation, cleavage of procaspases-3, -8, and 9, truncation of proapoptotic Bid, and release of cytochrome c from mitochondria. Tumor cells expressed constitutively active NF-kappaB and sensitization to TRAIL involved inhibition of NF-kappaB by curcumin. These findings suggest that combined curcumin/TRAIL chemo-immunotherapy may be a beneficial adjunct to the standard therapeutic regimens for prostate cancer.     

Influence of hypoxia on TRAIL-induced apoptosis in tumor cells

PURPOSE: Tumor hypoxia reduces the efficacy of radiotherapy, many types of chemotherapy, and tumor necrosis factor-alpha (TNF-alpha). TRAIL (TNF-alpha-related apoptosis-inducing ligand) is a ligand for death receptors of the TNF superfamily shown to be selectively toxic for tumor cells and thereby a promising antineoplastic tool. The impact of hypoxia on TRAIL-induced apoptosis was examined in this study. METHODS AND MATERIALS: Apoptosis induction and growth rates of various tumor cell lines under hypoxia were evaluated in vitro. Biologically effective induction of hypoxia was verified by determination of hypoxia-inducible factor-1 (HIF-1) activation. The efficacy of TRAIL- and radiation-induced apoptosis under different oxygen conditions was quantified in vitro. The impact of Bcl-2 on TRAIL-induced apoptosis under hypoxia or normoxia was evaluated by comparing cells expressing Bcl-2 with a vector control. RESULTS: Moderate hypoxia caused no growth retardation or apoptosis, but led to activation of HIF-1 as a prerequisite of hypoxic gene induction. Cellular responses to TRAIL differed considerably among the cell lines tested. Hypoxia reduced radiation-induced, but not TRAIL-induced, apoptosis in the tested cell lines. Hypoxia did not induce Bcl-2 expression. Bcl-2 had a minor impact on the efficacy of TRAIL-induced apoptosis. CONCLUSION: Taken together, the data indicate that TRAIL is clearly effective under conditions of proven hypoxia.     

5, 7-Dimethoxyflavone sensitizes TRAIL-induced apoptosis through DR5 upregulation in hepatocellular carcinoma cells

Purpose  

5, 7-dimethoxyflavone (DMF) has been reported to induce apoptosis in various cancer cells. The aim of this study was to examine whether DMF sensitizes human hepatocellular carcinoma (HCC) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and its mechanism.     

促进人胰腺癌Aspc1细胞凋亡

目的： 探讨 IL-27 基因对人胰腺癌Aspc1细胞凋亡的影响及其体内抗肿瘤作用。 方法： 重组载体PA317/IL-27转染Aspc1细胞,G418筛选稳定转染 IL-27 基因的Aspc1细胞（Aspc1/IL-27）。ELISA、细胞计数法和流式细胞术分别检测IL-27对Aspc1细胞IL-27表达、细胞增殖和MHC-Ⅰ类分子表达的影响。将Aspc1/IL-27、Aspc1/LXSN（稳定转染空质粒的Aspc1细胞）和Aspc1细胞接种于裸鼠右背部皮下,观察Aspc1细胞移植瘤的生长情况和小鼠的生存期;TUNEL法检测移植瘤细胞的凋亡,电镜观察移植瘤细胞的超微结构变化。 结果： 成功建立稳定转染PA317/IL-27载体的Aspc1/IL-27细胞株。Aspc1/IL-27细胞高表达IL-27,而Aspc1/LXSN和Aspc1细胞不表达IL-27（P<0.01）。PA317/IL-27载体转染不影响Aspc1细胞表面MHC-Ⅰ类分子的表达（P>0.05）。Aspc1/IL-27组裸鼠移植瘤生长速度明显慢于Aspc1/LXSN组及Aspc1组（P<0.05）,且生存期延长（P<0.05）。Aspc1/IL-27组移植瘤细胞凋亡率明显高于Aspc1/LXSN和Aspc1组\[（19.5±2.4）% vs（8.5±03）%、（9.1±0.8）%,P<0.01\]。 结论： IL-27 基因转染胰腺癌Aspc1细胞后通过诱导肿瘤细胞凋亡发挥抗肿瘤作用。     

Survivin downregulation by siRNA sensitizes human hepatoma cells to TRAIL-induced apoptosis

Survivin, an anti-apoptotic protein, is abundantly expressed in a variety of cancer cells, including hepatoma cells, resulting in the resistance of these cells to various apoptotic stimuli. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known to induce cancer cell-specific apoptosis, but hepatoma cells are resistant to TRAIL-induced apoptosis. In the present study, we have examined whether the downregulation of survivin by short interfering RNA (siRNA) promotes spontaneous or TRAIL-induced apoptosis in Huh-7 human hepatoma cells. Survivin siRNA transfection downregulated the expression of survivin in Huh-7 cells and reduced cell viability by 20% through inducing spontaneous apoptosis. TRAIL (1 to 2 ng/ml) only slightly induced apoptosis in Huh-7 cells; however, survivin siRNA transfection apparently enhanced TRAIL-induced apoptosis. These results suggest that the level of survivin is linked to the susceptibility of Huh-7 cells to TRAIL. It is possible that survivin downregulation by siRNA combined with TRAIL administration may provide a new therapeutic strategy against hepatoma.     

Simultaneous gene silencing of Bcl-2, XIAP and Survivin re-sensitizes pancreatic cancer cells towards apoptosis

Background  

Pancreatic ductal adenocarcinoma shows a distinct apoptosis resistance, which contributes significantly to the aggressive nature of this tumor and constrains the effectiveness of new therapeutic strategies. Apoptosis resistance is determined by the net balance of the cells pro-and anti-apoptotic "control mechanisms". Numerous dysregulated anti-apoptotic genes have been identified in pancreatic cancer and seem to contribute to the high anti-apoptotic buffering capacity. We aimed to compare the benefit of simultaneous gene silencing (SGS) of several candidate genes with conventional gene silencing of single genes.     

eNOS protects prostate cancer cells from TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anti-cancer agent because it induces apoptosis of most tumor cells with little or no effect on normal cells. In this study, we investigated the effect of TRAIL on human prostate normal and cancer cell lines, and found that the prostate cancer cell lines PC-3, ALVA-31, DU 145 and TSU-Pr1 were sensitive to TRAIL-induced apoptosis, while normal PrEC cells and cancer cell line LNCaP were resistant. No correlation was found between the sensitivity of cells to TRAIL and the expression of TRAIL receptors DR4 and DR5, and pro-apoptotic proteins Bax and Bak. However, LNCaP cells displayed a high Akt activity. Furthermore, we found that endothelial nitric oxide synthase (eNOS), one of the Akt substrates, was highly expressed in LNCaP but not in other cells. Inhibition of eNOS activity by NOS inhibitor sensitized LNCaP cells to TRAIL. Moreover, PC-3 cell clones stably expressing eNOS were resistant to TRAIL-induced apoptosis. Taken together, these results indicate that eNOS can regulate the sensitivity of prostate cancer cells to TRAIL, and down-regulation of eNOS activity may sensitize prostate cancer cells to TRAIL-based therapy.     

Chemotherapeutic agents enhance TRAIL-induced apoptosis in prostate cancer cells

PURPOSE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) family that preferentially kills tumor cells. In this study, we sought to determine whether chemotherapeutic agents augment TRAIL-induced cytotoxicity in human prostate cancer cells, and whether this sensitivity can be blocked by overexpression of bcl-2. METHODS: Prostate cancer cells, PC3 and LNCaP, were treated with TRAIL alone, drug alone or a combination of both for 24 h. Cytotoxicity was determined by DNA fragmentation and clonogenic survival assay. RESULTS: Treatment with the conventional chemotherapeutic agents cisplatin (2 and 5 microg/ml), etoposide (10 microM and 20 microM) and doxorubicin (30 and 60 n M) dramatically augmented TRAIL-induced apoptosis in LNCaP and PC3 cells. TRAIL-induced apoptosis was partially abrogated by overexpression of bcl-2 in these two cell lines when it was used in combination with the above agents. Similar results were obtained using clonogenic survival assays where bcl-2 overexpression was also found to marginally protect against TRAIL- and chemotherapy-induced cell killing.CONCLUSIONS: This study demonstrates that combination treatment of prostate cancer cells with TRAIL and chemotherapeutic agents overcomes their resistance by triggering caspase activation. This greater than additive effect of cotreatment with TRAIL and chemotherapy may provide the basis for a new therapeutic approach to induce apoptosis in otherwise resistant cancer cells.