Inhibition of melanoma tumor growth in vivo by survivin targeting

A role of apoptosis (programmed cell death) in tumor formation and growth was investigated by targeting the apoptosis inhibitor survivin in vivo. Expression of a phosphorylation-defective survivin mutant (Thr(34)-->Ala) triggered apoptosis in several human melanoma cell lines and enhanced cell death induced by the chemotherapeutic drug cisplatin in vitro. Conditional expression of survivin Thr(34)-->Ala in YUSAC2 melanoma cells prevented tumor formation upon s.c. injection into CB.17 severe combined immunodeficient-beige mice. When induced in established melanoma tumors, survivin Thr(34)-->Ala inhibited tumor growth by 60-70% and caused increased apoptosis and reduced proliferation of melanoma cells in vivo. Manipulation of the antiapoptotic pathway maintained by survivin may be beneficial for cancer therapy.     

A molecular chaperone glucose-regulated protein 94 blocks apoptosis induced by virus infection

The hepatitis C virus (HCV) E2 protein has been shown to block apoptosis and has been suggested to facilitate persistent infection of the virus. Here, we report that the anti-apoptotic activity of E2 is mediated by activation of nuclear factor kappa B (NF-kappaB) that directs expression of survival gene products such as tumor necrosis factor (TNF-alpha) receptor-associated factor 2 (TRAF2), X-chromosome-linked inhibitor of apoptosis protein (XIAP), FLICE-like inhibitory protein (FLIP), and survivin. Increased levels of these proteins were observed in HCV-infected cells and a cell line producing HCV E2 protein. The activation of NF-kappaB was mediated by HCV-E2-induced expression of the molecular chaperone glucose-regulated protein 94 (GRP94). Overexpression of GRP94 alone resulted in expression of anti-apoptotic proteins and blocked apoptosis induced by tumor-necrosis-related apoptosis-inducing ligand (TRAIL). Interestingly, increased levels of GRP94 were observed in cells supporting HCV proliferation that originated from liver tissues from HCV patients. Moreover, small interfering RNA (siRNA) knock-down of GRP94 nullified the anti-apoptotic activity of HCV E2. CONCLUSION: These data indicate that HCV E2 blocks apoptosis induced by HCV infection and the host immune system through overproduction of GRP94, and that HCV E2 plays an important role in persistent HCV infection.     

Prognostic value of survivin,X‐linked inhibitor of apoptosis protein and second mitochondria‐derived activator of caspases expression in advanced non‐small‐cell lung cancer patients

Background and objective: Survivin and X‐linked inhibitor of apoptosis protein (XIAP) in vitro mediate cancer cell survival and chemoresistance. Second mitochondria‐derived activator of caspases (Smac), an antagonist of XIAP, has been shown in vitro to increase chemosensitivity. This study examined the prognostic value of survivin, XIAP and Smac in advanced non‐small‐cell lung cancer (NSCLC) patients treated with cisplatin‐containing chemotherapy. Methods: Semi‐quantitative RT‐PCR was used to measure survivin, XIAP and Smac mRNA expression in transbronchial biopsy tumour specimens from 72 patients with advanced NSCLC before commencing chemotherapy. Outcome measures were response to chemotherapy, progression‐free survival (PFS) and overall survival (OS). Results: Low expression of survivin was associated with good response to chemotherapy (P = 0.028). No association was found between XIAP and Smac expression levels and response to chemotherapy (P = 0.224 and P = 0.088, respectively). Patients with low survivin expression or high Smac expression had significantly longer PFS (P = 0.012 and P = 0.029, respectively) and OS (P = 0.007 and P = 0.031, respectively) compared with patients with high expression of survivin or low expression of Smac. XIAP expression was not correlated with PFS or OS. Additionally, PFS and OS in patients with performance status of 0 or 1 and stage IIIB were significantly longer than PFS and OS in patients with performance status (PS) of 2 and stage IV disease. Multivariate Cox regression analyses demonstrated that survivin and clinical stage were independent predictors for PFS and OS. Smac was an independent prognostic factor for OS, but not for PFS. Conclusions: Our findings suggest that the expression levels of survivin and Smac, but not XIAP, predict the survival of patients with advanced NSCLC treated with chemotherapy.     

The Akt/Mcl-1 pathway plays a prominent role in mediating antiapoptotic signals downstream of the B-cell receptor in chronic lymphocytic leukemia B cells

Sustained engagement of the B-cell receptor (BCR) increases apoptosis resistance in chronic lymphocytic leukemia (CLL) B cells, whereas transient stimulation usually has an opposite effect. The antiapoptotic BCR signal has been associated with prolonged activation of the PI3K/Akt and MEK/ERK pathways, which are key regulators of survival and proliferation in various cell types. To further define the relative contribution of the Akt and ERK kinases in regulating CLL B-cell survival, we introduced constitutively active mutants of Akt and MEK in primary CLL B cells and evaluated changes in the expression of relevant pro- and antiapoptotic proteins. Sustained activation of Akt resulted in increased leukemic cell viability and increased expression of the antiapoptotic proteins Mcl-1, Bcl-xL, and X-linked inhibitor of apoptosis protein (XIAP), thus largely recapitulating the effects of sustained BCR stimulation. Constitutively active MEK2 also up-regulated XIAP, but did not show a significant impact on leukemic cell survival. Down-regulation of Mcl-1 by siRNA treatment induced rapid and potent apoptosis in CLL B cells and blocked the antiapoptotic effect of sustained BCR stimulation, whereas down-regulation of Bcl-xL and XIAP did not affect leukemic cell viability. These data demonstrate that Akt and Mcl-1 are major components of a survival pathway that can be activated in CLL B cells by antigen stimulation.     

汉防己甲素抑制PANC-1细胞增殖作用的机制探讨

目的：探讨汉防己甲素对人胰腺癌细胞株PANC-1细胞增殖的影响及其相关机制。方法：采用MTT法观察汉防己甲素对人胰腺癌细胞株PANC-1细胞增殖的影响;流式细胞术检测其对PANC-1细胞周期及凋亡的影响;实时定量PCR检测细胞周期和凋亡相关基因P21^cip/waf1、cdc25A、c-Myc、E2F1和survivin mRNA水平的变化;Western blot检测P21^cip/waf1蛋白水平的改变。结果：MTT结果显示汉防己甲素对PANC-1细胞增殖抑制作用呈明显的剂量和时间依赖性。经汉防己甲素处理后,流式细胞术检测到明显的G1期细胞阻滞和细胞凋亡。实时定量PCR结果显示,汉防己甲素处理早期,P21^cip/waf1mRNA水平明显增加,而cdc25A、c-Myc、2F1、survivin mRNA水平均下降。Western blot结果表明P21^cip/waf1蛋白水平在汉防己甲素处理早期升高。结论：我们的研究表明汉防己甲素能有效的抑制人胰腺癌细胞株PANC-1细胞增殖。该过程可能通过上调21^cip/waf1mRNA和蛋白的表达,下调cdc25A、c-Myc、E2F1mRNA的表达,致使PANC-1细胞G1期阻滞;也可能通过下调survivin mRNA的表达,诱导PANC-1细胞凋亡。     

The Role of cIAP1 and XIAP in Apoptosis Induced by Tumor Necrosis Factor Alpha in Esophageal Squamous Cell Carcinoma Cells

Background

The inhibitor of apoptosis protein (IAP) family are reported to play important roles in cancer cells evading apoptosis. However, the significance of their expression in human esophageal squamous cell carcinoma (ESCC) cells remains uncertain.

Aims

The present study aimed to investigate the role of the IAP family members in tumor necrosis factor-α (TNF-α)-induced apoptosis of human ESCC cells.

Methods

Five human ESCC cell lines were pretreated with TNF-α, cycloheximide (CHX, protein synthesis inhibitor), epoxomicin (proteasome inhibitor). Apoptosis assay and protein study with Western blot testing were conducted. Knockdown experiments with IAP siRNA were conducted, and the effect on cell apoptosis was analyzed.

Results

Significant apoptosis was induced in five ESCC cell lines by TNF-α plus CHX stimulation, but not when treated with TNF-α or CHX alone. The protein expression levels of cIAP1 and XIAP were decreased by treatment with TNF-α in the presence of CHX, and the degree of cIAP1 and XIAP expression decrease was correlated with sensitivity to TNF-α plus CHX-induced apoptosis. Epoxomicin suppressed TNF-α plus CHX-induced degradation of survivin, cIAP1, and XIAP, in addition to apoptosis. A caspase inhibitor (z-VAD-fmk) suppressed TNF-α plus CHX-induced apoptosis, but did not suppress degradation of survivin, cIAP1, and XIAP. Furthermore, cIAP1 or XIAP siRNA transfected cells underwent apoptosis in response to treatment with TNF-α alone. Double knockdown of both genes resulted in further increased apoptosis.

Conclusion

cIAP1 and XIAP play an essential role in the resistance of ESCC cells against apoptosis.

     

着丝粒蛋白F在非小细胞肺癌组织中的表达及其对A549细胞生物学行为的影响研究

背景非小细胞肺癌(NSCLC)占肺癌的85%左右,由于易错失最佳手术切除时机,且放化疗效果不佳,导致生存率偏低。而着丝粒蛋白F(CENPF)是在多种癌症中高表达的分子,探讨其对肺癌细胞生物学行为的影响,将有助于NSCLC的靶向治疗。目的探讨CENPF在NSCLC组织中的表达及其对A549细胞生物学行为的影响。方法收集2018年10月—2019年10月于西安医学院第一附属医院行外科切除手术的26例NSCLC患者的癌组织、癌旁组织标本。采用Western blotting法检测NSCLC组织、癌旁组织中CENPF表达水平。取生长良好的对数期A549细胞,将其分为对照组、阴性对照组、CENPF抑制组,其中对照组A549细胞不进行转染处理,阴性对照组、CENPF抑制组A549细胞使用Lipofectamine 2000转染试剂分别转染阴性对照(NC)-干扰性小核糖核酸(si RNA)、CENPF-si RNA。采用实时荧光定量聚合酶链式反应(q PCR)法检测三组A549细胞中CENPF m RNA表达水平,细胞计数试剂盒8(CCK-8)法检测A549细胞增殖情况[吸光度(OD)值],流式细胞仪检测A549细胞凋亡情况、A549细胞周期,Western blotting法检测A549细胞中CENPF、细胞周期蛋白D1(cyclin D1)、B淋巴细胞瘤-2基因相关X蛋白(Bax)表达水平。结果 NSCLC组织中CENPF表达水平高于癌旁组织(P <0.05)。CENPF抑制组A549细胞中CENPF m RNA表达水平低于对照组、阴性对照组(P<0.05)。CENPF抑制组A549细胞OD值低于对照组、阴性对照组(P<0.05)。CENPF抑制组A549细胞凋亡率高于对照组、阴性对照组(P <0.05)。CENPF抑制组G0/G1期A549细胞所占比例高于对照组、阴性对照组,S期、G2/M期A549细胞所占比例低于对照组、阴性对照组(P <0.05)。CENPF抑制组A549细胞中CENPF、cyclin D1表达水平低于对照组、阴性对照组,Bax表达水平高于对照组、阴性对照组(P <0.05)。结论 CENPF在NSCLC组织中表达升高,其可通过影响cyclin D1、Bax表达水平来影响A549细胞周期进展及增殖、凋亡过程;下调其表达可将A549细胞周期阻滞于G0/G1期,抑制细胞增殖并诱导其凋亡。     

Resveratrol inhibits proliferation, induces apoptosis, and overcomes chemoresistance through down-regulation of STAT3 and nuclear factor-kappaB-regulated antiapoptotic and cell survival gene products in human multiple myeloma cells

Whether resveratrol, a component of red grapes, berries, and peanuts, could suppress the proliferation of multiple myeloma (MM) cells by interfering with NF-kappaB and STAT3 pathways, was investigated. Resveratrol inhibited the proliferation of human multiple myeloma cell lines regardless of whether they were sensitive or resistant to the conventional chemotherapy agents. This stilbene also potentiated the apoptotic effects of bortezomib and thalidomide. Resveratrol induced apoptosis as indicated by accumulation of sub-G(1) population, increase in Bax release, and activation of caspase-3. This correlated with down-regulation of various proliferative and antiapoptotic gene products, including cyclin D1, cIAP-2, XIAP, survivin, Bcl-2, Bcl-xL, Bfl-1/A1, and TRAF2. In addition, resveratrol down-regulated the constitutive activation of AKT. These effects of resveratrol are mediated through suppression of constitutively active NF-kappaB through inhibition of IkappaBalpha kinase and the phosphorylation of IkappaBalpha and of p65. Resveratrol inhibited both the constitutive and the interleukin 6-induced activation of STAT3. When we examined CD138(+) plasma cells from patients with MM, resveratrol inhibited constitutive activation of both NF-kappaB and STAT3, leading to down-regulation of cell proliferation and potentiation of apoptosis induced by bortezomib and thalidomide. These mechanistic findings suggest that resveratrol may have a potential in the treatment of multiple myeloma.     

nitric oxide triggers the phosphatidylinositol 3-kinase/Akt survival pathway in insulin-producing RINm5F cells by arousing Src to activate insulin receptor substrate-1

Mechanisms involved in the protective action of nitric oxide (NO) in insulin-producing cells are a matter of debate. We have previously shown that pharmacological inhibition of c-Src cancels the antiapoptotic action of low and sustained concentrations of exogenous NO. In this study, using insulin-producing RINm5F cells that overexpress Src either permanently active (v-Src) or dominant negative (dn-Src) forms, we determine that this tyrosine kinase is the principal mediator of the protective action of NO. We also show that Src-directed activation of insulin receptor substrate-1, phosphatidylinositol 3-kinase (PI3K), Akt, and Bad phosphorylation conform a substantial component of the survival route because pharmacological inhibition of PI3K and Akt canceled the antiapoptotic effects of NO. Studies performed with the protein kinase G (PKG) inhibitor KT-5823 revealed that NO-dependent activation of c-Src/ insulin receptor substrate-1 is not affected by PKG activation. By contrast, Akt and Bad activation are partially dependent on PKG activation. Endogenous production of NO after overexpression of endothelial nitric oxide synthase in RINm5F cells mimics the effects produced by generation of low amounts of NO from exogenous diethylenetriamine/NO. In addition, we found that NO produces c-Src/PI3K- and PKG-dependent activation of ERK 1/2. The MAPK kinase inhibitor PD 98059 suppresses NO-dependent protection from DNA fragmentation induced by serum deprivation. The protective action of low and sustained concentration of NO is also observed in staurosporine- and Taxol-induced apoptosis. Finally, NO also protects isolated rat islets from DNA fragmentation induced by serum deprivation. These data strengthen the notion that NO production at physiological levels plays a role in protection from apoptosis in pancreatic beta-cells.     

非诺贝特联用顺铂对人肺癌A549细胞抑制和凋亡的实验研究

目的探讨过氧化物酶体增殖因子激活受体仪激动剂非诺贝特联用顺铂对体外培养的人肺癌A549细胞增殖和凋广的影响及可能机制。方法采用MTF法分别检测单用非诺贝特、顺铂和非诺贝特联用顺铂对肺癌A549细胞的生长抑制情况。采用Hoechest染色观察非诺贝特联用顺铂对肺癌A549细胞的生长抑制作用。流式细胞术检测联合用药对A549细胞的凋亡诱导作用。RT—PCR法检测联合用药对A549细胞中caspase－3、survivinmRNA的表达变化情况。结果非诺贝特对肺癌A549细胞有抑制作用,呈现时间剂量关系。联合用药组作用肺癌A549细胞48h后比单用药组的细胞抑制作用强（P〈0．05）。形态学观察及流式细胞术结果显示联合用药组A549凋亡细胞数目多于单药组,RT—PCR结果提示联合用药组较单用药组上调caspase－3mRNA的表达,下调stirvivin mRNA的表达。结论非诺贝特与顺铂联用可以增强顺铂杀伤肺癌A549细胞作用,其机制可能与上调caspase－3的表达和下调survivin的表达有关。     

Tumstatin185～191对人肺腺癌细胞的抑制作用及其与蛋白激酶B和细胞外调节蛋白激酶活性的关系

目的 观察Tumstatin185～191对肺腺癌细胞增生与凋亡的影响,探讨其与蛋白激酶B(Akt)及细胞外调节蛋白激酶(ERK)活性的关系.方法 以人肺腺癌细胞株A549为研究对象,分别给予不同浓度的Tumstatin185～191、顺铂及Tumstatin185～191联合顺铂进行干预,以四唑盐比色法测定A549细胞的增殖情况;流式细胞仪检测分析A549细胞凋亡的变化;Western blot检测细胞内磷酸化的Akt(p-Akt)及磷酸化的ERK(p-ERK)蛋白表达水平.结果 Tumstatin185～191对A549细胞的半数抑制浓度(IC_(50))为73.7μmol/L,顺铂对A549细胞的IC_(50)为5.2μmol/L,当顺铂与20μmol/L或40μmol/L的Tumstatin185～191联合应用时,顺铂对A549细胞的IC_(50)分别下降为3.5 μmol/L和1.4μmol/L;两药联合使用时,A549细胞的早期凋亡率为(19.34±0.97)%,较顺铂[(12.5±2.1)%]和Tumstatin185～191单用[(9.6±1.6)%]的早期凋亡率显著增加(F=5.74,P<0.01);Western blot检测显示p-Akt及p-ERK在未经干预的A549细胞内均呈高表达状态,Tumstatin185～191可显著抑制p-Akt及p-ERK的表达,顺铂则无明显影响;Tumstatin185～191与顺铂联合使用并不增加Tumstatin185～191对p-Akt及p-ERK的抑制效应.结论 Tumstatin185～191单药或联合顺铂对于A549细胞均有显著抑制作用,Tumstatin185～191能够增加A549细胞对顺铂的敏感性;Tumstatin185～191可能通过下调A549细胞内p-Akt及p-ERK水平发挥其抑制细胞生长、促进细胞凋亡的作用.     

Regulation of survivin function by Hsp90

Pathways controlling cell proliferation and cell survival require flexible adaptation to environmental stresses. These mechanisms are frequently exploited in cancer, allowing tumor cells to thrive in unfavorable milieus. Here, we show that Hsp90, a molecular chaperone that is central to the cellular stress response, associates with survivin, an apoptosis inhibitor and essential regulator of mitosis. This interaction involves the ATPase domain of Hsp90 and the survivin baculovirus inhibitor of apoptosis repeat. Global suppression of the Hsp90 chaperone function or targeted Abmediated disruption of the survivin-Hsp90 complex results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis, and cell cycle arrest with mitotic defects. These data link the cellular stress response to an antiapoptotic and mitotic checkpoint maintained by survivin. Targeting the survivin-Hsp90 complex may provide a rational approach for cancer therapy.     

金葡菌对U937细胞XIAP和cIAP1/2表达的影响

目的探讨X 相关凋亡抑制蛋白（XIAP）和cIAP1/2在金黄色葡萄球菌（简称金葡菌）诱导的人巨噬细胞系U937细胞凋亡中的作用。方法采用Western 印迹分析检测金葡菌感染不同时间后XIAP和cIAP1/2的表达水平;预先用不同浓度的Embelin（XIAP抑制剂）处理U937细胞60 min,观察金葡菌感染30 min后U937细胞的凋亡情况。结果随着感染时间的延长,XIAP和cIAP1/2的表达逐渐下降。加入XIAP的抑制剂Embelin后,U937细胞的凋亡率随着Embelin的浓度增加而逐渐升高。结论XIAP和cIAP1/2参与了金葡菌诱导的U937细胞凋亡过程。Embelin通过特异性地抑制XIAP表达增加金葡菌诱导的U937细胞凋亡率。     

X-linked inhibitor of apoptosis protein is an important regulator of vascular endothelial growth factor-dependent bovine aortic endothelial cell survival

Vascular endothelial growth factor (VEGF) is a critical regulator of endothelial cell biology and vascular function. Chronic VEGF treatment has been shown to inhibit tumor necrosis factor-induced apoptosis in endothelial cells. However, the mechanism for this cell survival is unclear. Interestingly, VEGF also enhances the expression of X-linked inhibitor of apoptosis (XIAP), a well-established antiapoptotic factor. XIAP has been shown to suppress apoptosis by blocking caspase activity in cancer cells, but it remains under studied in the endothelium. Therefore, we hypothesized that VEGF affects important endothelial functions, such as apoptosis and cell migration, by regulating XIAP expression and downstream caspase activity. To test this hypothesis, caspase activity, apoptosis, and cell migration were assessed following XIAP overexpression or depletion in bovine aortic endothelial cells. Much like VEGF treatment, ectopic expression of XIAP blocked tumor necrosis factor-induced apoptosis. Surprisingly, the mechanism was caspase-independent. In addition, XIAP-associated cell survival was the result of enhanced nitric oxide (NO) production, and XIAP was partially localized in caveolae. In these lipid rafts, XIAP interacted with a regulator of NO production, caveolin-1, via a binding motif (FtFgtwiY, where the bold letters represent aromatic amino acids) in the baculoviral IAP repeat-3 domain. Endothelial NO synthase binding to caveolin-1 was competitively inhibited by XIAP, suggesting that XIAP acts as a modulator of NO production by releasing endothelial NO synthase from caveolin-1. Further studies showed that endothelial cell migration was also controlled by XIAP-dependent NO. Taken together, these results suggest that XIAP plays a novel role in endothelial cells, interacting with caveolin-1 and acting as a regulator of vascular antiatherogenic function.     

Novel nonclassical antifolate, 2-[N-(2´-Hydroxyethyl)amino]methyl-3H-quinazolin-4-one, with a potent antineoplastic activity toward leukemia cells

This study was aimed to investigate the therapeutic potential of novel nonclassical antifolate, 2-[N-(2′-Hydroxyethyl)ami-no]methyl-3H-quinazolin-4-one (HEAMQ), toward human promonocytic U937 and murine lymphoblastic L1210 cell lines. The antiproliferative activity of HEAMQ was determined by MTT assay and its effects on cell cycle progression and apoptosis were studied by flow cytometry, and by immunoblots, respectively. In addition, combination chemotherapy of HEAMQ with cisplatin and temozolomide under in vitro and in vivo conditions was tested. HEAMQ showed concentration- and time-dependent cytotoxicity toward U937 and L1210 cells. It induced G2/M arrest that in U937 cells was associated with a marked decrease in the protein expressions of cyclin A, cyclin B, and cyclin-dependent kinase Cdk1. HEAMQ-induced apoptosis was accompanied with up-regulation of the protein expression of Bax and down-regulation of the protein expression of Bcl-2, Mcl-1, XIAP, and survivin, resulting in cytochrome c release and activation of caspases. Inhibitors of JNK (SP600125) and p38 MAPK (SB203580) suppressed HEAMQ-induced apoptosis and G2/M phase arrest, attenuated the activation of Bax, and blocked down-regulation of Bcl-2, XIAP and survivin in HEAMQ-treated U937 cells. In addition, combinations of HEAMQ with cisplatin and temozolomide resulted in synergistic inhibition of cell growth, producing long-term survivors of L1210 tumor-bearing mice. In conclusion, these results indicate that HEAMQ antineoplastic activity toward leukemia cells is associated with cell cycle arrest and apoptosis. The in vivo studies further confirmed the antitumor activity of HEAMQ and highlighted that this agent could be used to further increase therapeutic efficacies of traditional chemotherapeutic agents. Keywords: nonclassical antifolate, quinazolinone, antineoplastic, apoptosis, cell cycle arrest, synergism.     

Targeting X-linked inhibitor of apoptosis protein inhibits pancreatic cancer cell growth through p-Akt depletion

AIM: To determine whether lentivirus-mediated shRNA targeting the X-linked inhibitor of apoptosis protein (XIAP) gene could be exploited in the treatment of pancreatic cancer.METHODS: Human pancreatic cancer cells Panc-1, Mia-paca2, Bxpc-3 and SW1990, infected with lentivirus, were analyzed by real-time polymerase chain reaction (PCR). Western blotting was used to examine XIAP protein levels, survivin and p-Akt to confirm the result of real-time PCR and determine the possible mechanism. The 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to measure IC₅₀ to determine chemosensitivity to the chemotherapeutic drugs 5-fluorouracil (5-FU) and gemcitabine. A colony assay, MTT assay and a tumorigenicity experiment were used to study cell proliferation in vitro and in vivo. Caspase-3/7 activity, 4'',6-diamidino-2-phenylindole-staining and flow cytometric measurements were used to study apoptosis in SW1990 cells.RESULTS: XIAP proteins were found to be differentially expressed among pancreatic cancer cell lines Panc-1, Mia-paca2, Bxpc-3 and SW1990. Data of real-time PCR and Western blotting showed that XIAP was reduced persistently and markedly by lentivirus-mediated shRNA. Downregulation of XIAP by transfection with XIAP shRNA resulted in decreased p-Akt expression. XIAP shRNA also inhibited the growth of pancreatic cancer cells in vitro and in vivo, enhanced drug-induced apoptosis and increased chemosensitivity to 5-FU and gemcitabine. Results also suggest that inhibition of XIAP and subsequent p-Akt depletion may have an anti-tumor effect through attenuating the ability of cancer cells to survive.CONCLUSION: Lentivirus-mediated gene therapy is an attractive strategy in the treatment of pancreatic cancer and justifies the use of lentivirus in pancreatic cancer gene therapy studies.     

Caspase-independent cell death in AML: caspase inhibition in vitro with pan-caspase inhibitors or in vivo by XIAP or Survivin does not affect cell survival or prognosis

Survivin and XIAP, members of the protein family known as the inhibitors of apoptosis, interfere with the activation of caspases, called the "cell death executioners." We examined Survivin (n = 116) and XIAP (n = 172) expression in primary acute myeloid leukemia (AML) blasts and assessed the impact of their expression on prognosis. They were detected in all samples analyzed. However, no correlation was observed with cytogenetics, remission attainment, or overall survival of patients with AML. To investigate the importance of caspases in chemotherapy-induced apoptosis in AML, we treated OCI-AML3 cells with Ara-C, doxorubicin, vincristine, and paclitaxel, which induced caspase cleavage and apoptosis. Blocking of caspase activation by pan-caspase inhibitor abolished poly(adenosine diphosphate [ADP]-ribose) polymerase cleavage and DNA fragmentation but did not prevent chemotherapy-induced cell death and did not inhibit, or only partially inhibited, mitochondrial release of cytochrome c, Smac, apoptosis-inducing factor (AIF), or loss of mitochondrial membrane potential. Caspase inhibition also did not protect AML blasts from chemotherapy-induced cell death in vitro. These results suggest that expression levels of Survivin or XIAP have no prognostic impact in AML patients. Although anticancer drugs induced caspase cleavage and apoptosis, cell killing was caspase independent. This may partially explain the lack of prognostic impact of XIAP and Survivin and may suggest caspase-independent mechanisms of cell death in AML.