氟达拉滨与TRAIL联用诱导肺癌细胞凋亡及 其作用机制

目的：研究氟达拉滨（Fludarabine）在低浓度下与TRAIL联合处理是否能够诱导人肺癌A549细胞发生凋亡,并探讨其作用机制。方法：实验设对照组、Fludarabine组、TRAIL组、Fludarabine和TRAIL联合组,CCK-8法检测Fludarabine与TRAIL联合处理时对A549细胞及人脐静脉内皮细胞（HUVEC,人正常细胞作为对照）增殖活力的影响,流式细胞术检测A549细胞凋亡情况,Western blot法检测凋亡相关蛋白Survivin、Bcl-2、Bcl-xl、caspase-8、caspase-3、JNK和p38的表达情况。结果：25、50 μmol/LFludarabine或50 ng/mL TRAIL单独处理时对A549及HUVEC细胞增殖活力均无明显影响（P均>0.05）,两者联合处理后对A549细胞增殖活力抑制率分别为30.9%和44.9%。Fludarabine或TRAIL单独使用时对A549细胞凋亡无明显影响,联合处理时可使A549细胞凋亡率达到89.3%。与对照组、Fludarabine组和TRAIL组相比,Fludarabine与TRAIL联合处理可使A549细胞中Survivin、Bcl-2、Bcl-xl表达降低（P均<0.05）,促进caspase-8及caspase-3的激活,并能促进JNK和p38的磷酸化（P均>0.05）。结论：Fludarabine与TRAIL联合处理可促进A549细胞凋亡。     

Genistein sensitizes TRAIL-resistant human gastric adenocarcinoma AGS cells through activation of caspase-3

The cytotoxic effect of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is limited in some cancer cells, including AGS gastric adenocarcinoma cells. However, treatment with TRAIL in combination with subtoxic concentrations of genistein sensitizes TRAIL-resistant AGS cells to TRAIL-mediated apoptosis. Combined treatment with genistein and TRAIL-induced chromatin condensation and sub-G1 phase DNA content. These indicators of apoptosis are correlated with the activation of death receptors (DR5) and induction of caspase-3 activity, which results in the cleavage of poly(ADP-ribose)polymerase. Both the cytotoxic effect and apoptotic characteristics induced by combined treatment were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role of caspase-3 in the observed cytotoxic effect. These results indicate that caspase-3 is a key regulator of apoptosis in response to combined genistein and TRAIL in human gastric adenocarcinoma AGS cells through the activation of DR5 and mitochondrial dysfunction.     

Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitor adaphostin proceeds through a RAF-1/MEK/ERK- and AKT-dependent process

Effects of the tyrphostin tyrosine kinase inhibitor adaphostin (NSC 680410) have been examined in human leukemia cells (Jurkat, U937) in relation to mitochondrial events, apoptosis, and perturbations in signaling and cell cycle regulatory events. Exposure of cells to adaphostin concentrations > or =0.75 microM for intervals > or =6 h resulted in a pronounced release of cytochrome c and AIF, activation of caspase-9, -8, and -3, and apoptosis. These events were accompanied by the caspase-independent downregulation of Raf-1, inactivation of MEK1/2, ERK, Akt, p70S6K, dephosphorylation of GSK-3, and activation of c-Jun-N-terminal kinase (JNK) and p38 MAPK. Adaphostin also induced cleavage and dephosphorylation of pRb on CDK2- and CDK4-specific sites, as well as the caspase-dependent downregulation of cyclin D1. Inducible expression of a constitutively active MEK1 construct markedly diminished adaphostin-induced cytochrome c and AIF release, JNK activation, and apoptosis in Jurkat cells. Ectopic expression of Raf-1 or constitutively activated (myristolated) Akt also significantly attenuated adaphostin-induced apoptosis, but protection was less than that conferred by enforced activation of MEK. Lastly, antioxidants (e.g., L-N-acetylcysteine; L-NAC) opposed adaphostin-mediated mitochondrial dysfunction, Raf-1/MEK/ERK downregulation, JNK activation, and apoptosis. However, in contrast to L-NAC, enforced activation of MEK failed to block adaphostin-mediated ROS generation. Together, these findings demonstrate that the tyrphostin adaphostin induces multiple perturbations in signal transduction pathways in human leukemia cells, particularly inactivation of the cytoprotective Raf-1/MEK/ERK and Akt cascades, that culminate in mitochondrial injury, caspase activation, and apoptosis. They also suggest that adaphostin-related oxidative stress acts upstream of perturbations in these signaling pathways to trigger the cell death process.     

Reconstitution of caspase-3 confers low glucose-enhanced tumor necrosis factor-related apoptosis-inducing ligand cytotoxicity and Akt cleavage.

PURPOSE AND EXPERIMENTAL DESIGN: We have previously observed that glucose deprivation enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death as well as caspase activation (caspase-3, -9, and -8) in human prostate adenocarcinoma DU-145 cells. In this study, we used caspase-3-deficient MCF-7 breast cancer cells to examine the possible role of caspase-3 in glucose deprivation-enhanced TRAIL cytotoxicity. RESULTS: Combined glucose deprivation and 200 ng/ml TRAIL treatment markedly induced cytotoxicity in caspase-3 cDNA transfected cells (MCF-7/casp-3) but not in control vector transfected cells (MCF-7/vector). We also observed that the level of Akt, an antiapoptotic protein, was reduced by treatment with TRAIL in MCF-7/casp-3 cells but not in MCF-7/vector cells. The reduction of Akt by TRAIL was promoted in the absence of glucose in MCF-7/casp-3 cells. However, pretreatment with 20 micro M Z-LEHD-FMK, a caspase-9 inhibitor, protected MCF-7/casp-3 cells from the combinatorial treatment of TRAIL and glucose deprivation-induced cytotoxicity. This compound also prevented the reduction of Akt level during the combinatorial treatment. Moreover, this Akt reduction was not inhibited by treatment with MG-132, a proteosome inhibitor. Data from site-directed mutagenesis show that Akt was cleaved at amino acid 108, but not 119, during treatment with TRAIL and glucose deprivation. CONCLUSIONS: Our results suggest that caspase-3 is involved in the reduction of Akt level, and its involvement is mediated through caspase-9 activation. The reduction of Akt level is also due to cleavage of Akt rather than degradation of Akt.     

Tamoxifen-induced apoptosis of rat C6 glioma cells via PI3K/Akt, JNK and ERK activation

To elucidate the mechanism of TAM treatment on gliomas, we hypothesised that PI3K/Akt and MAPK signaling pathway may play important roles on TAM-induced apoptosis in C6 glioma cells. Our results demonstrated that TAM induced apoptosis of C6 glioma cells in a dose-dependent manner. The activation of AKT significantly decreased in a time-dependent manner in response to TAM treatment, JNK was transiently activated, and subsequently decreased activation and kept stable level, whereas ERK evidenced sustained activations in response to the drug treatment. The inhibition of PI3K/Akt and JNK both accelerated and enhanced TAM-induced apoptosis and ERK inhibition apparently exerted negative effect on apoptosis. We also observed that PI3K/Akt had intimate association with JNK and ERK activation in TAM-induced apoptosis. These findings may provide strategies for the molecularly targeted therapy in malignant gliomas.     

Benzyl isothiocyanate (BITC) induces apoptosis in ovarian cancer cells in vitro

Advanced ovarian cancer (OC) is not curable by surgery alone and chemotherapy is essential for its treatment. Isothiocyanates have been shown to inhibit carcinogen-induced tumorigenesis in animal models, yet no efforts have been made to determine their therapeutic potential in OC. In the present study, we investigated the mechanism of the anti-proliferative and apoptotic activity of benzyl isothiocyanate (BITC) in OC. BITC inhibited the proliferation of OC cells and induced apoptosis in OC cells. Apoptosis was induced by a strong activation of caspase-3 and -9, and cleavage of PARP-1. However, caspase-8 was not activated by BITC. Cytotoxic effects of BITC were reversed by the inhibition caspase-3 and -9 specific inhibitors. BITC showed a concentration dependent decrease in the levels of Bcl-2 with a concomitant increase in Bax levels. In addition, BITC activated proapoptotic signaling by phosphorylation JNK1/2 and p38 while simultaneously inhibiting survival signaling mediated by ERK1/2 and Akt phosphorylation in a dose-dependent manner. While JNK inhibitor SP600125 and p38 inhibitor SB203580, abolished the cytotoxic effect of BITC, MEK inhibitor, PD98059 and PI3 kinase inhibitor, LY294002 failed to show such reversal indicating a critical role played by JNK1/2 and p38 signaling in apoptosis induced by BITC. In summary, our studies demonstrate that BITC inhibits proliferation of OC cells and induces apoptosis via caspase-9 and -3 pathways. BITC inhibits ERK1/2 and Akt survival signaling while simultaneously activating pro-apoptotic p38 and JNK1/2. Therefore, BITC can be potentially developed as a therapeutic agent to treat OC.     

Roles of JNK, p38 and ERK mitogen-activated protein kinases in the growth inhibition and apoptosis induced by cadmium

Cadmium (Cd), a human carcinogen, can induce apoptosis in various cell types. Three major mitogen-activated protein kinases (MAPKs), c-JUN N-terminal kinase (JNK), p38 and extracellular signal-regulated kinase (ERK), have been shown to regulate apoptosis. In this study we explore the ability of Cd to activate JNK, p38 and ERK, including their effects on Cd-mediated growth inhibition and apoptosis in a human non-small cell lung carcinoma cell line, CL3. The kinase activity of JNK was induced dose-dependently by 30-160 microM CdCl(2). High cytotoxic doses of Cd (130-160 microM) markedly activated p38, but low Cd doses did not. Conversely, the activities of ERK1 and ERK2 were decreased by low cytotoxic doses of Cd (相似文献   

The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells

Dietary phytochemicals have been shown to be protective against various types of cancers. However, the precise underlying protective mechanisms are poorly understood. In the present study, we report that treatment of A549 cells with quercetin resulted in a dose-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 1.2 +/- 0.8, 6.3 +/- 0.9, 16.5 +/- 1.5, 36.4 +/- 2.6 and 42.5 +/- 5.8% on treatment with 0.1% dimethylsulfoxide, 14.5, 29.0, 43.5 and 58.0 micro M quercetin, respectively. Concomitantly, quercetin treatments led to a 1.1-, 1.1-, 2.5- and 3.5-fold increase in Bax. Similar elevations were also observed in Bad, which increased 1.1-, 2.1-, 2.2- and 2.3-fold, respectively, as compared with control. While Bcl-2 was decreased by 30%, Bcl-x(L) was elevated in a dose-dependent fashion. Quercetin also induced the cleavage of caspase-3, caspase-7 and PARP (poly ADP-ribose polymerase). While Akt-1 and phosphorylated Akt-1 were inhibited, the extracellular signal-regulated kinase (ERK) was phosphorylated following quercetin treatment in a dose-dependent fashion. Phosphorylation of ERK and c-Jun occurred at 3 h and was sustained over 14 h. Phosphorylation of MEK1/2 was increased in concordance with ERK activation. Quercetin-induced phosphorylation of c-Jun N-terminal kinase (JNK) and cleavage of caspase-3 occurred 6 h after quercetin exposure and before cleavage of caspase-7 and PARP was detected. Inhibition of MEK1/2 but not PI-3 kinase, p38 kinase or JNK abolished quercetin-induced phosphorylation of c-Jun, cleavage of caspase-3 and -7, cleavage of PARP and apoptosis. Inhibition of caspase activation completely blocked quercetin-induced apoptosis. Expression of constitutively activated MEK1 in A549 cells led to activation of caspase-3 and apoptosis. The results suggest that in addition to inactivation of Akt-1 and alteration in the expression of the Bcl-2 family of proteins, activation of MEK-ERK is required for quercetin-induced apoptosis in A549 lung carcinoma cells.     

亚砷酸诱导肺腺癌A549细胞凋亡及对MAPK／ERK信号通路的影响

目的探讨亚砷酸对肺腺癌A549细胞凋亡、MAPK／ERK信号通路的影响及其作用机制。方法体外培养肺腺癌A549细胞,实验组采用不同浓度(1、3、6 μmol／L)亚砷酸处理,对照组加入等体积的二甲基亚砜(DMSO),MTT法检测细胞增殖率,FMC法检测细胞周期分布与细胞凋亡率,Hoechst 33342观察凋亡小体,Western blotting检测Bcl 2、Bax、p53、Caspase 3及MAPK／ERK信号通路相关蛋白表达水平,RT PCR检测PCNA、CDK2、CyclinA1表达量。结果实验组肺腺癌A549细胞增殖率、凋亡率、凋亡小体数量随着亚砷酸处理时间延长而降低,且存在剂量反应效应(P<005);Western blotting结果显示,实验组肺腺癌A549细胞中Bax、p53、Caspase 3、p JNK／JNK及p38蛋白水平高于对照组,而Bcl 2、p ERK／ERK蛋白含量低于对照组,均呈剂量依赖性,差异均有统计学意义(P<005);RT PCR结果显示实验组肺腺癌A549细胞PCNA、CDK2、CyclinA1表达量低于对照组,均呈剂量依赖性,差异均有统计学意义(P<005)。结论亚砷酸可以通过下调细胞周期基因水平、上调细胞凋亡相关因子含量、降低MAPK／ERK信号传导,来诱导肺腺癌A549细胞凋亡。     

Sulforaphane enhances TRAIL-induced apoptosis through the induction of DR5 expression in human osteosarcoma cells

Sulforaphane (SFN), a naturally occurring isothiocyanate, is an attractive agent because of its potent anticancer effects. SFN suppresses the proliferation of various cancer cells in vitro and in vivo. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is also one of the most promising candidates for cancer therapeutics owing to its ability to selectively induce apoptosis in tumor cells. In this study, we report that SFN enhances TRAIL-induced apoptosis in human osteosarcoma cells, Saos2 and MG63. The apoptosis induced by co-treatment with SFN and TRAIL was markedly blocked by a dominant negative form of the TRAIL receptor or caspase inhibitors. The combined use of SFN and TRAIL effectively induced Bid cleavage and the activation of caspases 8, 10, 9 and 3 at ineffective concentrations for each agent. SFN upregulated the expression of death receptor 5 (DR5), a receptor for TRAIL, at mRNA and protein levels in a dose-dependent manner. In addition, the SFN-mediated sensitization to TRAIL was reduced by DR5 siRNA, suggesting that the sensitization was at least partially mediated through the induction of DR5 expression. Furthermore, SFN sensitized TRAIL-induced apoptosis in a p53-independent manner. On the other hand, SFN neither induced DR5 protein expression or enhanced TRAIL-induced apoptosis in normal human peripheral blood mononuclear cells. Thus, combined treatment with SFN and TRAIL might be a promising therapy for osteosarcoma.     

JNK/FOXO3信号通路介导黄连素促人肺腺癌PC-9细胞凋亡的研究

  目的  探讨黄连素（berberine,Ber）诱导肺腺癌PC-9细胞凋亡及c-Jun氨基末端激酶（c-Jun N-terminal kinase,JNK）/转录因子叉头蛋白3（forkhead box protein O3,FOXO3）信号通路的作用机制。  方法  实验采用完全随机化的分组方法,分为对照组、Ber组（30、60 μM）,分别检测各组PC-9细胞活力值、凋亡率、ROS含量、caspase 3活性、线粒体膜电位,以及JNK/FOXO3通路和凋亡相关蛋白含量的变化。SP600125特异性抑制JNK（磷酸化）激活后,Ber（0、60 μM）处理细胞24 h,重复上述检测。  结果  Ber有效抑制PC-9细胞活力,促进细胞凋亡（P < 0.05）,显著降低PC-9细胞线粒体膜电位,增加ROS含量和caspase 3活性（P < 0.05）,并呈浓度依赖效应;Western blot检测结果显示Ber上调p-JNK、FOXO3和Bax的含量（P < 0.05）,下调p-FOXO3和Bcl-2的含量（P < 0.05）;SP600125特异性抑制JNK激活后,拮抗Ber下调p-FOXO3含量及其促PC-9细胞凋亡作用（P < 0.05）。  结论  Ber可有效抑制肺腺癌PC-9细胞活力、促进细胞凋亡和氧化应激损伤,作用机制可能与上调p-JNK、FOXO3含量和抑制FOXO3磷酸化有关。      

TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt

The majority of leukaemic cells are resistant to apoptosis induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we show that sublethal concentrations of arsenic trioxide (ATO) specifically enhanced TRAIL-induced apoptosis in leukaemic but not in other tumour cell lines. The combination of ATO and TRAIL synergistically enhanced cleavage of caspase-8, which was blocked by the caspase inhibitor IETD.fmk as well as in cells deficient for caspase-8, suggesting a requirement for the death-inducing signalling complex. Arsenic trioxide led to increased cell surface expression of DR5 (death receptor 5), inhibition of the serine/threonine kinase Akt and downregulation of the short isoform of FLIP (FLICE-inhibitory protein, FLIPS). Inhibition of the phosphatidylinositol 3 kinase (PI3K) was equally efficient in sensitising leukaemic cells to TRAIL with similar effects on DR5 and FLIPS expression, suggesting that ATO may in part act through inhibition of the PI3K/Akt signalling pathway. These results indicate that the enhancement in TRAIL-mediated apoptosis induced by ATO is due to alteration in the levels of multiple components and regulators of the death receptor-mediated pathway. These findings offer a promising and novel strategy involving a combination of TRAIL and ATO, or more specific Akt inhibitors in the treatment of various haematopoietic malignancies.     

青藤碱诱导肺癌NCI-H460细胞凋亡及其机制的实验研究

目的:探究青藤碱(SIN)诱导肺癌NCI-H460细胞凋亡的作用机制。方法:四甲基偶氮唑蓝(MTT)法测定SIN对肺癌NCI-H460细胞生长的影响;Western blot测定Bcl-2,Bax蛋白的表达;用SIN、PI3K/Akt和MAPK/ERK信号通路抑制剂干预NCI-H460细胞,流式细胞术检测细胞的凋亡率。结果:SIN对肺癌NCI-H460细胞生长有抑制作用,呈时间、浓度依赖性;SIN可以使NCI-H460细胞Bcl-2表达减低,Bax增强;SIN与PI3K/Akt和MAPK/ERK信号通路抑制剂联用后可协同增加NCI-H460细胞凋亡(P<0.05)。结论:SIN可以抑制肺癌NCI-H460细胞的生长,能改变其Bax,Bcl-2蛋白的表达,与PI3K/Akt和MAPK/ERK信号通路抑制剂联用可协同发挥促进肺癌细胞凋亡作用,可望成为新的肺癌治疗药物。     

Constitutively active Akt1 protects HL60 leukemia cells from TRAIL-induced apoptosis through a mechanism involving NF-kappaB activation and cFLIP(L) up-regulation.

TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL. We found that parental (PT) HL60 cells were very sensitive to a 6 h incubation in the presence of TRAIL and died by apoptosis. In contrast, AR cells were resistant to TRAIL concentrations as high as 2 microg/ml for 24 h. Two pharmacological inhibitors of PI3K, Ly294002 and wortmannin, restored TRAIL sensitivity of AR cells. AR cells stably overexpressing PTEN had lower Akt1 activity and were sensitive to TRAIL. Conversely, PT cells stably overexpressing a constitutive active form of Akt1 became TRAIL resistant. TRAIL activated caspase-8 but not caspase-9 or -10 in HL60 cells. We did not observe a protective effect of Bcl-X(L) or Bcl-2 against the cytotoxic activity of TRAIL, even though TRAIL induced cleavage of BID. There was a close correlation between TRAIL sensitivity and intranuclear presence of the p50 subunit of NF-kappaB. Higher levels of the FLICE inhibitory protein, cFLIP(L), were observed in TRAIL-resistant cells. Both the cell permeable NF-kappaB inhibitor SN50 and cycloheximide lowered cFLIP(L)expression and restored sentivity of AR cells to TRAIL. Our results suggest that Akt1 may be an important regulator of TRAIL sensitivity in HL60 cells through the activation of NF-kappaB and up-regulation of cFLIP(L) synthesis.     

Interferon-α sensitizes human gastric cancer cells to TRAIL-induced apoptosis via activation of the c-CBL-dependent MAPK/ERK pathway

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family that induces apoptosis in cancer cells. However, gastric cancer cells are insensitive to TRAIL. In the present study, we show that pretreatment with IFN-α enhanced TRAIL-induced apoptosis of gastric cancer MGC803 cells. IFN-α up-regulated death receptor 5 (DR5) expression and down-regulated survivin expression. Furthermore, extracellular-regulated protein kinase (ERK1/2) activation was induced by IFN-α, and a combination of IFN-α and TRAIL led to further activation of ERK1/2. Inhibition of the MAPK/ERK signaling pathway partially reversed apoptosis, as well as the expression patterns of DR5 and survivin. Moreover, the expression of the c-casitas B-lineage lymphoma (c-Cbl) family was down-regulated by IFN-α. Transfection of c-Cbl suppressed IFN-α-induced ERK activation. These results indicate that IFN-α enhances TRAIL-induced apoptosis in gastric cancer cells at least partially via downregulation of c-Cbl, and subsequent up-regulation of the MAPK/ERK pathway.     

2-Chloro-2'-deoxyadenosine-induced apoptosis in T leukemia cells is mediated via a caspase-3-dependent mitochondrial feedback amplification loop

2-Chloro-2'-deoxyadenosine (CdA; cladribine) is a chemotherapeutic agent used in the treatment of certain leukemias. However, the signalling events that govern CdA-mediated cytotoxicity in leukemia cells remain unclear. We show here that CdA treatment caused Jurkat human T leukemia cells to die via apoptosis in a dose- and time-dependent fashion. Bcl-2 overexpression protected Jurkat T leukemia cells from CdA-induced apoptosis and loss of mitochondrial transmembrane potential (Delta Psi m). Furthermore, mitochondria that were isolated from Jurkat T leukemia cells and then exposed to CdA showed a loss of Delta Psi m, indicating that CdA directly compromised outer mitochondrial membrane integrity. CdA treatment of Jurkat T leukemia cells resulted in the activation of caspase-3, -8, and -9, while inhibition of these caspases prevented the CdA-induced loss of Delta Psi m, as well as DNA fragmentation. In addition, caspase-3 inhibition prevented caspase-8 activation while caspase-8 inhibition prevented caspase-9 activation. Death receptor signalling was not involved in CdA-induced apoptosis since cytotoxicity was not affected by FADD-deficiency or antibody neutralization of either Fas ligand or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Taken together, these data suggested that CdA-induced apoptosis in Jurkat T leukemia cells was mediated via a caspase-3-dependent mitochondrial feedback amplification loop. CdA treatment also increased p38 mitogen-activated protein (MAPK) and extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in Jurkat T leukemia cells. Although ERK1/2 inhibition did not affect CdA-mediated cytotoxicity, inhibition of p38 MAPK had an enhancing effect, which suggested a cytoprotective function for p38 MAPK. Agents that inhibit p38 MAPK might therefore increase the effectiveness of CdA-based chemotherapy.     

Adriamycin Sensitizes Adriamycin-Resistant HL-60/ADR Cells to TRAIL-Mediated Apoptosis

OBJECTIVE To study whether an adriamycin-resistant cell line(HL-60/ADR) can be sensitized by adriamycin(ADR) to TRAIL-mediated apoptosis.METHODS The mRNA levels of the TRAIL receptor and apoptosis-related signaling molecules involved in the TRAIL-mediated apoptotic pathway were measured by RT-PCR.The protein levels of apoptotic-related signaling molecules involved in the TRAIL-mediated apoptotic pathway and processed caspase-3,caspase-9,and caspase-8 were measured by Western blots.Apoptosis was assessed by flow cytometry.Mitochondrial membrane potential was analyzed by DiOC6(3) staining.Cytotoxicity was determined by the colorimetric MTT viability/ proliferation assay.RESULTS Treatment with a combination of TRAIL and subtoxic concentrations of ADR resulted in synergistic cytotoxicity and apoptosis for both the parental HL-60 and the HL-60/ADR cells.For HL-60,there was a 5-fold potentiation and synergy in cytotoxicity for TRAIL and for HL-60/ADR,cytotoxicity to TRAIL was potentiated 6-fold with ADR.Adriamycin treatment modestly up-regulated TRAIL-R2(DR5),but had no effect on the expression of Fas-associated death domain,c-FLIP,Bcl-2,Bcl-xL,Bax,and IAP family members(cIAP-1,cIAP-2,XIAP,and survivin).The protein levels of pro-caspase-8 and pro-caspase-3 were not affected by ADR,whereas pro-caspase-9 and Apaf-1 were up-regulated.Combined treatment with TRAIL and ADR resulted in activation of caspase-9 and caspase-3,but there was no detectable processing of caspase-8 beyond the background levels.There was signif icant depolarization of the mitochondrial membrane by the combined treatment of both cell lines and it was more pronounced in the parental HL-60 cell line.The combined treatment with TRAIL and ADR resulted in 42.6% of the HL-60/ADR cells undergoing DNA fragmentation,whereas treatment with either ADR or TRAIL alone resulted in 5.46% and 21.3% DNA fragmented cells,respectively.Similar results were obtained with the HL-60 cells.CONCLUSION These fi ndings demonstrate that ADR can still signal ADR-resistant tumor cells,resulting in the modifi cation of the TRAIL-mediated signaling pathway and apoptosis.