Early apoptosis and cell death induced by ATX-S10Na ( Ⅱ)-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

AIM: To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (Ⅱ).METHODS: HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting.RESULTS: Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-XL,were decreased in Bax- and p53-independent manner.CONCLUSION: Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizingphotosensitizer ATX-S10Na (Ⅱ) are mediated by p53-Bax network and Iow levels of Bcl-2 and Bcl-XL proteins.Our results might help in formulating new therapeutic approaches in photedynamic therapy.     

Early apoptosis and cell death induced by ATX-S10Na （ Ⅱ ）-mediated photodynamic therapy are Bax- and p53-dependent in human colon cancer cells

AIM： To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na （Ⅱ）. METHODS： HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin Ⅴ assays, transmission electron microscopy assays, caspase assays and western blotting. RESULTS： Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, were decreased in Bax- and p53-independent manner. CONCLUSION： Our results indicate that eady apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizing photosensitizer ATX-S10Na （Ⅱ） are mediated by p53- Bax network and low levels of Bcl-2 and Bcl-xL proteins. Our results might help in formulating new therapeutic approaches in photodynamic therapy.     

Triptolide sensitizes AML cells to TRAIL-induced apoptosis via decrease of XIAP and p53-mediated increase of DR5

Acute myeloid leukemia (AML) cells are relatively resistant to tumor necrosis factor –related apoptosis-inducing ligand (TRAIL). We previously reported that triptolide, a potent anticancer agent from a Chinese herb, decreases XIAP in leukemic cells. We evaluated the combination of triptolide and TRAIL and found synergistic promotion of apoptosis in AML cells. XIAP-overexpressing U937 cells (U937XIAP) were more resistant to TRAIL than U937neo cells, and inhibition of XIAP with the small-molecule inhibitor 1396-11 enhanced TRAIL-induced apoptosis, implying XIAP as a resistance factor in AML. Furthermore, triptolide increased DR5 levels in OCI-AML3, while the DR5 increase was blunted in p53-knockdown OCI-AML3 and p53-mutated U937 cells, confirming a role for p53 in the regulation of DR5. In support of this finding, disruption of MDM2-p53 binding with subsequent increase in p53 levels by nutlin3a increased DR5 levels and sensitized OCI-AML3 cells to TRAIL. The combination of 1396-11 plus nutlin3a plus TRAIL was more effective than either the 1396-11 and TRAIL or nutlin3a and TRAIL combinations in OCI-AML3 cells, further supporting the role of triptolide as a sensitizer to TRAILinduced apoptosis in part by independent modulation of XIAP expression and p53 signaling. Thus, the combination of triptolide and TRAIL may provide a novel strategy for treating AML by overcoming critical mechanisms of apoptosis resistance.     

Inactivating mutations of KILLER/DR5 gene in gastric cancers.

BACKGROUND & AIMS: The KILLER/death receptor (DR)5 has been identified as a potent inducer of apoptosis, and mapped to chromosome 8p21-22, showing frequent allelic loss in gastric cancer. The p53-induced apoptosis is an important biological process to prevent the development of cancer, and is mediated in part by expression of KILLER/DR5 only in cells with wild-type p53 protein, but not in those lacking p53 function. The aim of this study was to determine whether genetic alterations of KILLER/DR5 could be involved in the tumorigenesis of gastric cancer. METHODS: We analyzed the genetic alterations of KILLER/DR5 and p53 in 43 gastric cancers and the loss of function of KILLER/DR5 mutants, detected in this study. RESULTS: We found 3 KILLER/DR5 missense mutations (7%), and 2 of them showed allelic loss in the remaining allele. Interestingly, all the mutants inhibit apoptotic cell death in transfection studies. We also found 6 p53 mutations (14%). Interestingly, the tumors containing the KILLER/DR5 mutation did not carry the p53 mutation. CONCLUSIONS: These results suggest that inactivation of KILLER/DR5 caused by mutations of KILLER/DR5 may be one of the possible escaping mechanisms against KILLER/DR5-mediated apoptosis and that inactivating mutation of KILLER/DR5 may contribute to the development or progression of a subset of gastric cancers.     

Regulation of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in Burkitt's lymphoma cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in sensitive cells and may be suitable for novel anti-cancer therapies aimed at inducing apoptosis via the activation of TRAIL receptors on malignant cells. Here we have characterized the TRAIL sensitivity of a panel of Burkitt's lymphoma (BL) cell lines. Overall, 5/12 BL cell lines and 1/2 lymphoblastoid cell lines were sensitive to TRAIL-induced apoptosis, although only one BL cell line approached the sensitivity of Jurkat cells, a widely used model for TRAIL-induced apoptosis. Whereas, 4/5 of the Epstein-Barr virus (EBV)-negative cell lines were TRAIL sensitive, only 1/7 EBV-positive BL cell lines were TRAIL sensitive. However, isogenic BL cell lines with different EBV status were not differently sensitive to TRAIL, indicating that EBV is not a major determinant of TRAIL sensitivity. All cell lines expressed the death receptor (DR)5 TRAIL receptor, whereas expression of DR4 was more variable. Differences in the expression of downstream signalling molecules [Fas-associated death domain protein (FADD), caspase 8] and inhibitors [decoy receptor 1 (DcR1), cellular FLICE-like inhibitory protein (c-FLIP)] did not correlate with TRAIL sensitivity. Therefore, a subset of BL cell lines are sensitive to TRAIL-induced apoptosis, however, the molecular mechanism that determines responsiveness remains to be identified.     

错配修复基因MLH1激活p53和线粒体凋亡通路参与雌激素诱导的结肠癌细胞凋亡

临床和流行病学研究显示雌激素替代治疗可降低绝经后妇女的结直肠癌发生风险。前期研究发现雌激素能上调结肠癌细胞的错配修复(MMR)基因MLH1表达,在MLH1基因缺失的结肠癌细胞中再表达MLH1能明显增强雌激素诱导的细胞凋亡。目的:探讨MLH1参与雌激素诱导结肠癌细胞凋亡所涉及的信号通路以及p53及其相关基因在此凋亡通路中的作用。方法:以含人野生型MLH1(hMLH1)全长cDNA的质粒转染MLH1基因缺失的人结肠癌细胞株HCT116。以转染空质粒的HCT1 16细胞作为对照,在有或无雌激素作用的条件下,采用电泳法检测凋亡DNA Ladder,蛋白质印迹法检测p53等凋亡相关蛋白表达。结果:转染hMLH1后,10~(-8)mol/L雌二醇(E_2)能明显诱导HCT116细胞凋亡。转染hMLH1并经E_2处理的HCT116细胞(D组)与经E_2处理但未转染hMLH1的HCT116细胞(B组)相比,其caspase-3、caspase-9、p53、Bax、胞质细胞色素C蛋白表达均显著增强,D组上述蛋白表达亦均高于转染hMLH1但未经E_2处理的HCT116细胞(C组)。结论:MMR基因MLH1主要通过激活p53和线粒体凋亡通路参与雌激素诱导的人结肠癌细胞株HCT116凋亡。     

Chemical sensitization and regulation of TRAIL-induced apoptosis in a panel of B-lymphocytic leukaemia cell lines

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) effectively kills tumour cells but not normal cells. We investigated TRAIL sensitivity and the TRAIL-induced apoptosis signalling pathway in a panel of B-lymphocytic leukaemia cell lines. Depending upon TRAIL sensitivity, leukaemia cells could be divided into three groups: highly sensitive, moderately sensitive and resistant. TRAIL receptor-2 (DR5) plays an important role in transducing apoptosis signals. DR5 was internalized into the cytoplasm where it recruited FAS-associated death domain protein (FADD) under TRAIL stimulation in both sensitive and resistant cells. However, the active form of caspase-8 was recruited to FADD and only sensitive cells showed increased caspase-8 activity upon TRAIL stimulation. The caspase-8 specific inhibitor, Z-IETD, impaired caspase-8 activation and completely abrogated TRAIL-induced apoptosis. These results suggest that TRAIL resistance in B-lymphocytic leukaemia cells is due to negative regulation at the level of caspase-8 activation and that caspase-8 activation is an indispensable process in TRAIL-induced apoptosis. However, FADD-like interleukin-1 beta-converting enzyme inhibitory protein (c-FLIPL) was similarly expressed and down-regulated after TRAIL stimulation in both sensitive and resistant cells. Interestingly, in some cell lines, TRAIL sensitivity and caspase-8 activity was enhanced or restored with the treatment of cycloheximide (CHX). In addition, X-linked inhibitor of apoptosis (XIAP) levels decreased significantly and rapidly following treatment with CHX. Down-regulation of XIAP may be responsible for enhancement or restoration of TRAIL sensitivity after CHX treatment in B-lymphocytic leukaemia cells.     

Actinomycin D renders cultured synovial fibroblasts susceptible to tumour necrosis factor related apoptosis-inducing ligand (TRAIL)-induced apoptosis

OBJECTIVE: To investigate the expression of tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors in cultured synovial fibroblasts from rheumatoid arthritis (RA) and osteoarthritis (OA) patients, and to examine their susceptibility to TRAIL-induced apoptosis in the presence or absence of metabolic inhibitors. METHODS: The expression of TRAIL receptors in synovial fibroblasts was examined by Western blot and immunohistochemistry. Expression of TRAIL-receptor 1 (TRAIL-R1), FLICE-inhibitory protein (Fas-associating protein with death domain-like interleukin-1-converting enzyme), and Bcl-2 was assessed by Western blot. Synovial cell viability was measured by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay (XTT), and apoptosis was determined both by DNA content analysis after propidium iodide staining and Annexin V stain. RESULTS: TRAIL-R1 was constitutively expressed on cultured synovial fibroblasts from RA and OA, however, expression of TRAIL-R2 and TRAIL-R3 was not observed by immunohistochemistry and Western blot. Cultured synovial fibroblasts were resistant to apoptosis by TRAIL alone, but combined treatment of TRAIL with actinomycin D (ActD: 200 ng/mL), cycloheximide (CHX: 10 microg/mL), or proteasome inhibitor (MG132: 20 microM) induced apoptosis in a dose-dependent manner. The apoptosis was completely or partially inhibited by various caspase inhibitors, implicating an involvement of caspase pathway in TRAIL-induced apoptosis in the presence of these metabolic inhibitors. Expression of TRAIL-R1, FLIPL, and Bcl-2 did not account for the apoptosis by the combined treatment of TRAIL with ActD. CONCLUSIONS: Although TRAIL-R1 was constitutively expressed; cultured synovial fibroblasts were resistant to apoptosis by TRAIL. ActD, CHX, and MG132 rendered cultured synovial fibroblasts susceptible to TRAIL-induced apoptosis by a caspase-dependent mechanism. However, the exact mechanism of sensitization by these metabolic inhibitors remains to be determined.     

Induction of lysosomal membrane permeabilization by compounds that activate p53-independent apoptosis

The p53 protein activates cellular death programs through multiple pathways. Because the high frequency of p53 mutations in human tumors is believed to contribute to resistance to commonly used chemotherapeutic agents, it is important to identify drugs that induce p53-independent cell death and to define the mechanisms of action of such drugs. Here we screened a drug library (the National Cancer Institute mechanistic set; 879 compounds with diverse mechanisms of actions) and identified 175 compounds that induced caspase cleavage of cytokeratin-18 in cultured HCT116 colon cancer cells at <5 microM. Interestingly, whereas most compounds elicited a stronger apoptotic response in cells with functional p53, significant apoptosis was observed also in p53-null cells. A subset of 15 compounds showing weak or no dependence on p53 for induction of apoptosis was examined in detail. Of these compounds, 11 were capable of activating caspase-3 in enucleated cells. Seven such compounds with nonnuclear targets were found to induce lysosomal membrane permeabilization (LMP). Translocation of the lysosomal proteases cathepsin B and cathepsin D into the cytosol was observed after treatment with these drugs, and apoptosis was inhibited by pepstatin A, an inhibitor of cathepsin D. Apoptosis depended on Bax, suggesting that LMP induced a mitochondrial apoptotic pathway. We conclude that a large number of potential anticancer drugs induce p53-independent apoptosis and that LMP is a mediator of many such responses.     

Hepatitis B virus enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity by increasing TRAIL-R1/death receptor 4 expression

BACKGROUND/AIMS: Apoptosis by death receptors, such as Fas and tumor necrosis factor (TNF)-alpha receptor-1, play a significant role in the pathogenesis of hepatitis B virus (HBV)-infections. Although liver also expresses death receptors for TNF-related apoptosis-inducing ligand (TRAIL), information is lacking regarding the effects of HBV on apoptosis by TRAIL. Thus, the aims of this study were to examine the effects of HBV replication on TRAIL cytotoxicity. METHODS: Hep G2 and Hep G2.215 cells, the latter which is stably transfected with HBV, were employed for these studies. RESULTS: TRAIL-mediated cell killing was concentration-dependent and greater in Hep G2.2.15 cells at all doses as compared to the parent cell line, Hep G2 cells. Cell death by apoptosis was confirmed by demonstrating caspase activation and inhibition of cell killing by a caspase inhibitor, zVAD-fmk. TRAIL-R1/DR4 protein expression was enhanced in Hep G2.2.15 cells as compared to Hep G2 cells. Lamivudine treatment reduced TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression in Hep G2.2.15 cells. In Hep G2 cells transfected with the HBV-encoded X antigen (HBxAg), sensitivity to TRAIL-mediated apoptosis and TRAIL-R1/DR4 expression were both increased. CONCLUSIONS: TRAIL-induced apoptosis is enhanced by the level of HBV replication in human hepatocytes, in part, by HBxAg-dependent upregulation of TRAIL-R1/DR4.     

Casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells

ObjectiveTo assess if casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.MethodsHuman non-small-cell lung carcinoma cell lines H460, A549 and H157 were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cells death was examined by flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, -8 and -9 were measured via ELISA. Cellular fractionation was determined by flow cytometry to assess release of cytochrome c and the mitochondrial transmembrane potential. Bcl-2/Bcl-XL/XIAP/Bid/DR5 and DR4 proteins were analyzed using western blot.ResultsThe concentrations required for a 50% decrease in cell growth (IC₅₀) ranged from 1.8 to 3.2 μM. Casticin induced rapid apoptosis and triggered a series of effects associated with apoptosis by way of mitochondrial pathway, including the depolarization of the mitochondrial membrane, release of cytochrome c from mitochondria, activation of procaspase-9 and -3, and increase of DNA fragments. Moreover, the pan caspase inhibitor zVAD-FMK and the caspase-3 inhibitor zDEVD-FMK suppressed casticin-induced apoptosis. In addition, casticin induced XIAP and Bcl-XL down-regulation, Bax upregulation and Bid clearage. In H157 cell line, casticin increased expression of DR5 at protein levels but not affect the expression of DR4. The pretreatment with DR5/Fc chimera protein effectively attenuated casticin-induced apoptosis in H157 cells. No correlation was found between cell sensitivity to casticin and that to p53 status, suggesting that casticin induce a p53-independent apoptosis.ConclusionsOur results demonstrate that casticin induces caspase-mediated apoptosis via activation of mitochondrial pathway and upregulation of DR5 in human lung cancer cells.     

Down-regulation of Cbl-b by bufalin results in up-regulation of DR4/DR5 and sensitization of TRAIL-induced apoptosis in breast cancer cells

Purpose

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic agent that preferentially induces apoptosis in cancer cells. However, breast cancer cells are generally resistant to TRAIL. Bufalin is a major active ingredient of the traditional Chinese medicine ChanSu. The present study aimed to assess the synergistic effect of bufalin and TRAIL and elucidate the underlying mechanisms in breast cancer cells.

Methods

Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. The expression of proteins was assayed by flow cytometry and/or Western blotting. Transfection studies were used to determine the involvement of DR4, DR5 and Cbl-b in the synergistic effect of bufalin and TRAIL.

Results

MCF-7 and MDA-MB-231 cells were resistant to TRAIL. Both cell lines were dramatically sensitized to TRAIL-induced apoptosis by bufalin. Further experiments indicated that bufalin up-regulated DR4 and DR5, activated ERK, JNK and p38 MAPK and down-regulated Cbl-b. Blocking the up-regulation of DR4 and DR5 by siRNA rendered cells less sensitive to apoptosis induced by the combination of bufalin and TRAIL. Inhibition of the activation of ERK, JNK and p38 MAPK by specific inhibitors attenuated DR4 and DR5 up-regulation. Moreover, down-regulation of Cbl-b by shRNA led to stronger activation of ERK, JNK and p38 MAPK, more up-regulation of DR4 and DR5, and a stronger synergistic effect of bufalin and TRAIL.

Conclusions

Bufalin enhanced TRAIL-induced apoptosis by up-regulating the expression of DR4 and DR5. Bufalin-induced down-regulation of Cbl-b contributed to the up-regulation of DR4 and DR5, which might be partially mediated by the activation of ERK, JNK and p38 MAPK.     

TRAIL-mediated apoptosis requires NF-kappaB inhibition and the mitochondrial permeability transition in human hepatoma cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a wide range of malignant cells. However, several cancers, including human hepatoma, are resistant to TRAIL. In this study, we analyzed TRAIL-induced pro- and antiapoptotic signaling pathways in human hepatoma cells. Nuclear factor kappa B (NF-kappaB) was found to be a critical TRAIL-induced antiapoptotic factor in the PLC/PRF/5, HepG2, and Hep3B cell lines. TRAIL-induced NF-kappaB activation was preceded by IkappaBalpha kinase (IKK) activation and IkappaBalpha degradation and depended on TRAF2, NF-kappaB-inducing kinase (NIK), IKK1, and IKK2. Accordingly, inhibition of NF-kappaB by adenoviral dominant negative (dn) TRAF2, NIKdn, IKK1dn, IKK2dn, or IkappaBsr sensitized PLC/PRF/5 cells to rhTRAIL, resulting in 40% to 50% cell death after 48 hours as compared with <10% with rhTRAIL alone. Agonistic anti-TRAIL receptor 1 and anti-TRAIL receptor 2 antibodies or combinations of both were equally efficient in inducing apoptosis as rhTRAIL, indicating that decoy receptors did not contribute to resistance toward TRAIL under the conditions of our study. TRAIL-mediated apoptosis depended on FADD, caspase 8 and 3 as demonstrated by the ability of FADDdn, CrmA, and pharmacologic caspase inhibitors to prevent apoptosis. Confocal microscopy showed the onset of the mitochondrial permeability transition (MPT) 5 hours after rhTRAIL plus actinomycin D, which was followed by cytochrome c release. The MPT was critical for TRAIL-induced apoptosis as demonstrated by the ability of pharmacologic MPT inhibitors to completely protect PLC/PRF/5 cells. In conclusion, NF-kappaB prevents TRAIL-induced apoptosis in human hepatoma through a TRAIL-activated TRAF2-NIK-IKK pathway. Inhibition of NF-kappaB unmasks a TRAIL-induced apoptotic signaling cascade that involves FADD, caspase 8, the MPT, and caspase 3.     

Additive effect of Apo2L/TRAIL and Adeno-p53 in the induction of apoptosis in myeloma cell lines

OBJECTIVE: We have previously shown that Adenovirus-p53 (Ad-p53) is a potent inducer of apoptosis in myeloma cells expressing nonfunctional p53 and low levels of bcl-2 and that Apo2L/TRAIL is a potent inducer of apoptosis, independent of bcl-2. A study was designed to test the synergy between Ad-p53 and Apo2L/TRAIL in the induction of apoptosis in relation to the expression of DR4/DR5 and DcR1, in cells undergoing Ad-p53-induced apoptosis. METHODS: Replication deficient Ad-p53 and human recombinant Apo2L/TRAIL were used. Myeloma cells with mutated/w.t. p53 and varying expression of bcl-2 were used to test the effect of Ad-p53, Apo2L/TRAIL, or both, on apoptosis, measured by annexin V. RESULTS: Treatment with Ad-p53 resulted in a dose-dependent apoptosis concomitant with a dose-dependent increase in the expression of DR4/DR5 and a decrease in the expression of DcR1, in Ad-p53-sensitive cell lines. In these cells, addition of Apo2L/TRAIL to cells treated with Ad-p53 resulted in a dose-dependent increase in apoptosis. Myeloma cells resistant to Ad-p53 had high levels of DR4/DR5 and high levels of DcR1 and treatment with Ad-p53 did not reduce the expression of DcR1. Also, addition of Apo2L/TRAIL to Ad-p53 did not affect the level of apoptosis beyond the level of apoptosis observed with Apo2L/TRAIL alone. CONCLUSIONS: 1) Cotreatment with Ad-p53 and Apo2L/TRAIL resulted in additive apoptosis in myeloma cells expressing nonfunctional p53 and low levels of bcl-2. 2) Resistance to Ad-p53 or to the combination of Ad-p53 and Apo2L/TRAIL was not due to the lack of adenovirus receptor (CAR) or low expression of DR4/DR5 but rather due to the relatively high expression of DcR1 receptor.     

TRAIL induces apoptosis of chondrocytes and influences the pathogenesis of experimentally induced rat osteoarthritis

OBJECTIVE: To investigate whether TRAIL influences the pathogenesis of osteoarthritis (OA). METHODS: A recombinant adenoviral vector system (Ad-TRAIL) was used. Expression of TRAIL in a rat chondrocyte cell line (RCJ3.1C.18) and alterations in the expression of death and decoy receptors after Ad-TRAIL infection were measured by Western blot assay. To explore the underlying mechanism, Western blot assays (to detect caspase 8, poly[ADP-ribose] polymerase [PARP], and caspase 3 activation), mitochondrial membrane potential (DeltaPsim) measurement, Hoechst staining, and DNA electrophoresis were conducted. Next, expression of TRAIL and death and decoy receptors was examined by immunochemistry in primary cultured chondrocytes and on cartilage obtained from rats with experimentally induced OA. RESULTS: Ad-TRAIL infection induced expression of TRAIL in RCJ3.1C.18 cells, increased expression of death receptor 4 (DR4), and decreased expression of DR5 and decoy receptor 1 (DcR1). Ad-TRAIL, at doses of 10 and 100 multiplicities of infection, decreased the viability of chondrocytes 4 days after infection. Reduction of DeltaPsim, cytochrome c release, nuclear condensation, activation of caspase 3 and PARP, and DNA fragmentation proved the induction of apoptosis. Activation of caspase 8 was also observed. Ad-TRAIL also induced apoptosis in primary cultured chondrocytes, in which alterations in expression of TRAIL and death receptors were similar to those observed in RCJ3.1C.18 cells. Cartilage obtained from rats with experimentally induced OA showed increased expression of TRAIL and DR4 and decreased expression of DR5 and DcR1 compared with control cartilage. CONCLUSION: TRAIL induces chondrocyte apoptosis, and TRAIL-induced chondrocyte apoptosis may play a role in the pathogenesis of OA.     

5-FU增强TRAIL诱导的胃癌BGC823细胞凋亡的实验研究

目的 观察5-FU对TRAIL诱导的胃癌BGC823细胞凋亡的影响,明确死亡受体5(DR5)在5-FU和TRAIL诱导凋亡中的作用.方法 采用MTT法测定细胞活力、流式细胞仪检测细胞凋亡、免疫印迹检测蛋白表达.结果 TRAIL可导致BGC823细胞轻度的增殖抑制和少量的细胞凋亡.与单药TRAIL和5-FU相比,TRAIL联合5-FU对细胞的增殖抑制和诱导凋亡作用明显增强(P＜0.05).免疫印迹结果显示,TRAIL没有改变DR5的蛋白表达,而5-FU作用BGC823细胞48 h后,DR5蛋白表达上调(P＜0.05).TRAIL和5-FU联合作用后,DR5蛋白表达同样明显上调(P均＜0.05).结论 5-FU通过上调DR5蛋白表达提高了BGC823细胞对TRAIL的敏感性.     

Intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human multiple myeloma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma (MM) cells in vitro irrespective of refractoriness to dexamethasone and chemotherapy. Because clinical trials with this anticancer agent are expected shortly, we investigated the signaling pathway of TRAIL-induced apoptosis in MM. We detected rapid cleavage of caspases-8, -9, -3, and -6, as well as the caspase substrates poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45 (DFF45), but not caspase-10, upon TRAIL treatment in sensitive MM cells, pointing to caspase-8 as the apical caspase of TRAIL signaling in MM cells. These phenomena were not observed or were significantly delayed in TRAIL-resistant MM cells, suggesting that resistance may arise from inhibition at the level of caspase-8 activation. Higher levels of expression for various apoptosis inhibitors, including FLICE-inhibitory protein (FLIP), and lower procaspase-8 levels were present in TRAIL-resistant cells and sensitivity was restored by the protein synthesis inhibitor cycloheximide (CHX) and the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), which both lowered FLIP and cellular inhibitor of apoptosis protein-2 (cIAP-2) protein levels. Forced expression of procaspase-8 or FLIP antisense oligonucleotides also sensitized TRAIL-resistant cells to TRAIL. Moreover, the cell permeable nuclear factor (NF)-kappaB inhibitor SN50, which sensitizes TRAIL-resistant cells to TRAIL, also inhibited cIAP2 protein expression. Finally, CHX, BIM, and SN50 facilitated the cleavage and activation of procaspase-8 in TRAIL-resistant cells, confirming that inhibition of TRAIL-induced apoptosis occurs at this level and that these agents sensitize MM cells by relieving this block. Our data set a framework for the clinical use of approaches that sensitize MM cells to TRAIL by agents that inhibit FLIP and cIAP-2 expression or augment caspase-8 activity.     

Pretreatment with AT-101 enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of breast cancer cells by inducing death receptors 4 and 5 protein levels

Purpose

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has been shown to induce extrinsic pathway of apoptosis in many types of cancer cells. AT-101, an (?)-enantiomer of gossypol, is a potent anticancer agent that is shown to be an inhibitor of Bcl-2/Bcl-XL. In this study, we searched whether pretreatment with either of these drugs would result in the enhancement of apoptosis through induction of death receptors and activation of mitochondrial pathways within breast cancer cells.

Methods

Human breast cancer (MCF-7 and MDA-MB-231) and normal breast cells (MCF-10A) were treated with drugs alone/in combination/sequentially. XTT cell viability assay was used to evaluate cytotoxicity. For showing apoptosis, both DNA Fragmentation and caspase 3/7 activity measurements were done. ELISA and Western blot analysis were done to assess DR4 and DR5 protein levels. The expression levels of apoptotic proteins were assessed by human apoptosis antibody array.

Results

The sequential treatment of AT-101 followed by TRAIL resulted in significant synergistic cytotoxicity and apoptosis. Moreover, pretreatment of breast cancer cells with AT-101 and then with TRAIL caused enhancement of the expression levels of DR4 and DR5 in both cancer cell lines, suggesting that these cells were under strong apoptotic stimuli.

Conclusions

These findings all together, strongly suggest that pretreatment with AT-101 enhances TRAIL-induced death-inducing signaling complex resulting in the engagement of the mitochondrial pathway to apoptosis in breast cancer cells. These promising, preliminary results make AT-101 and TRAIL a novel combination treatment candidate for breast cancer.