Accelerated degradation of caspase-8 protein correlates with TRAIL resistance in a DLD1 human colon cancer cell line

The tumor-selective cytotoxic effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) makes TRAIL an attractive candidate as an anticancer agent. However, resistance to TRAIL poses a challenge in anticancer therapy with TRAIL. Therefore, characterizing the mechanisms of resistance and developing strategies to overcome the resistance are important steps toward successful TRAIL-mediated cancer therapy. In this study, we investigated mechanisms of acquired TRAIL resistance in a colon cancer DLD1 cell line. Compared with the TRAIL-susceptible DLD1 cell line, TRAIL-resistant DLD1/TRAIL-R cells have a low level of caspase-8 protein, but not its mRNA. Suppression of caspase-8 expression by siRNA in parental DLD1 cells led to TRAIL resistance. Restoration of caspase-8 protein expression by stable transfection rendered the DLD1/TRAIL-R cell line fully sensitive to TRAIL protein, suggesting that the low level of caspase-8 protein expression might be the culprit in TRAIL resistance in DLD1/TRAIL-R cells. Sequencing analysis of the caspase-8 coding region revealed a missense mutation that is present in both TRAIL-sensitive and TRAIL-resistant DLD1 cells. Subsequent study showed that the degradation of caspase-8 protein was accelerated in DLD1/TRAIL-R cells compared to parental DLD1 cells. Thus, accelerated degradation of caspase-8 protein is one of the mechanisms that lead to TRAIL resistance.     

Concentration-dependent positive and negative regulation of a MAP kinase by a MAP kinase kinase

There are at least three distinct MAP kinase signaling modules in mammalian cells, distinguished by the family of kinases (Erk, SAPK/JNK, or p38) that is ultimately activated. Many input signals activate multiple MAP kinase cascades, and the mechanisms that control the specificity of signal output are not well understood. We show that SEK1/MKK4, a MAP kinase kinase proposed to activate SAPK/JNK, is a very potent inhibitor of p54 SAPK beta/JNK3 both in vitro and in vivo if present at equimolar or higher ratios. In contrast SEK can activate SAPK when present in substoichiometric amounts, but this activation is slow, consistent with the rate-limiting step in activation being the dissociation of an inactive SEK:SAPK complex. The N-terminal unique region of SEK is both necessary and partially sufficient for inhibition of SAPK, and is also necessary for activation of SAPK by SEK in vitro. We have also used the p38 MAP kinase and its activator MKK6 to examine the regulatory relationships among different kinases involved in stress responses. We show using purified kinases that inhibitory activity is specific for the combination of SEK and SAPK: SEK can activate but not inhibit p38, and MKK6 can activate but not inhibit SAPK beta and p38. These results reveal a potential mechanism for regulating stress-activated kinases, adding to a growing body of evidence suggesting that MAP kinases are controlled by relatively stable interactions with their activators.     

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.     

结肠癌TRAIL耐药细胞株的建立及其耐药机制的初步研究

背景与目的以人结肠癌DLD1细胞株为载体,探讨恶性肿瘤获得性TRAIL基因耐药的可能机制。材料与方法用重组腺病毒介导的TRAIL基因(Ad/gTRAIL)反复处理人结肠癌DLD1细胞,得到耐药细胞群DLD1-TRAIL/R。通过MTT比色法和蛋白电泳,检测耐药细胞对Ad/gTRAIL杀伤作用的敏感性及其凋亡通路中信号分子的表达情况,分析其获得性耐药的可能机制。结果DLD1-TRAIL/R细胞对Ad/gTRAIL和重组TRAIL蛋白处理耐药,但对腺病毒介导的Bax基因处理仍然敏感。耐药细胞内Bcl-XL的表达明显升高,caspase-8的表达显著下降,未见明显的caspase-8活性裂解形式。结论人结肠癌DLD1细胞株经Ad/gTRAIL反复处理后产生特异性针对TRAIL基因的耐药,其发生机制可能与Bcl-XL表达上调和caspase-8表达下调有关。     

Silencing of prion protein sensitizes breast adriamycin-resistant carcinoma cells to TRAIL-mediated cell death

We investigated the relationship between the resistance to the proapoptotic action of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and cellular prion protein (PrPc) function, using the TRAIL-sensitive MCF-7 human breast adenocarcinoma cell line and two TRAIL-resistant sublines: 2101 and MCF-7/ADR. All of the cell lines tested expressed TRAIL-R1 and TRAIL-R2. TRAIL decoy receptors were not detected, suggesting that the resistance of 2101 and MCF-7/ADR cells, strongly expressing PrPc, to TRAIL-mediated cell death was independent from the expression of TRAIL receptors and death-inducing signaling complex formation. Down-regulation of PrPc by small interfering RNA increased the sensitivity of Adriamycin- and TRAIL-resistant cells to TRAIL, but not to epirubicin/Adriamycin. TRAIL-mediated apoptosis in PrPc knocked-down cells was associated with caspase processing, Bid cleavage, and Mcl-1 degradation. In addition, an increased sensitivity of apoptosis-resistant cells to TRAIL after PrPc silencing was not associated with the increased recruitment of receptors and intracellular signaling molecule to the death-inducing signaling complex. Bcl-2 expression was substantially decreased after PrPc knock-down but the levels of Bcl-X(L) and Mcl-1 were not affected. The down-regulation of Bcl-2 was concomitant with Bax delocalization. Our findings support the notion that silencing of PrPc facilitates the activation of proapoptotic Bax by down-regulation of Bcl-2 expression, thereby abolishing the resistance of breast cancer cells to TRAIL-induced apoptosis.     

Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated caspase-8 activation and apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new cytokine that was proposed to specifically induce apoptosis of cancer cells. In tumor cells that are resistant to the cytokine, subtoxic concentrations of chemotherapeutic drugs can restore the response to TRAIL. The present study further explores the mechanisms that determine tumor cell sensitivity to TRAIL by comparing four human colon carcinoma cell lines We show that colon cancer cell sensitivity to TRAIL-induced apoptosis and cytotoxicity correlates with the expression of the death receptors TRAIL-R1 and TRAIL-R2 at the cell surface, as determined by now cytometry, whereas the two decoy receptors TRAIL-R3 and TRAIL-R4 can be detected only in permeabilized cells. Clinically relevant concentrations of cisplatin and doxorubicin sensitize the most resistant colon cancer cell lines to TRAIL-induced cell death without modifying the expression nor the localization of TRAIL receptors in these cells. TRAIL induces the activation of procaspase-8 and triggers caspase-dependent apoptosis off colon cancer cells. Cytotoxic drugs lower the signaling threshold required for TRAIL-induced procaspase-8 activation. In turn, caspase-8 cleaves Bid, a BH3 domain-containing proapoptotic molecule of the Bcl-2 family and activates effector caspases. Together, these data indicate that chemotherapeutic drugs sensitize colon tumor cells to TRAIL-mediated caspase-8 activation and apoptosis.     

CD95 and TRAIL receptor-mediated activation of protein kinase C and NF-kappaB contributes to apoptosis resistance in ductal pancreatic adenocarcinoma cells.

The molecular alterations in tumour cells leading to resistance towards apoptosis induced by CD95 and TRAIL-receptors are not fully understood. We report here that the stimulation of the CD95- and TRAIL-resistant human pancreatic adenocarcinoma cell line PancTuI with an agonistic anti-CD95 antibody or TRAIL resulted in activation of protein kinase C and NF-kappaB. Inhibition of protein kinase C by G?6983 sensitized these cells to apoptotic challenges and strongly diminished activation of NF-kappaB by anti-CD95 and TRAIL. Similarly, inhibition of NF-kappaB by MG132 or by transient transfection with a dominant negative mutant of IkappaBalpha restored the responsiveness of PancTuI cells to both death ligands. In the CD95 and TRAIL-sensitive cell line Colo357 the induction of protein kinase C and NF-kappaB following activation of CD95 and TRAIL-R was very moderate compared with PancTuI cells. However, pre-incubation of these cells with PMA strongly reduced their apoptotic response to anti-CD95 and TRAIL. Taken together, we show that activation of protein kinase C operates directly in a death receptor-dependent manner in PancTuI cells and protect pancreatic tumour cells from anti-CD95 and TRAIL-mediated apoptosis by preventing the loss DeltaPsim and Cytochrome c release as well as by induction of NF-kappaB.     

The PI 3-kinase-Rac-p38 MAP kinase pathway is involved in the formation of signet-ring cell carcinoma

Signet-ring cell carcinoma is classified in poorly differentiated adenocarcinoma with an aggressive nature and a poor prognosis. We have shown that the activation of PI 3-kinase in highly differentiated adenocarcinomas induces loss of cell-cell contact and formation of vacuoles, giving phenotypes similar to those of signet-ring cell lines. SB203580, a potent p38 MAP kinase inhibitor, blocked this transition, and expression of an active form of MKK6 (MKK6DA), an activator of p38 MAP kinase, gave effects similar to those induced by expression of the active form of PI 3-kinase (BD110), although formation of large vacuoles was not induced. Activation of MKK3, another activator of p38 MAP kinase, was activated in native signet-ring carcinoma cell lines. Anchorage-independent growth of signet-ring cell lines was inhibited by LY294002 or SB203580. These results suggest that p38 MAP kinase is functioning downstream of PI 3-kinase in signaling of the malignant phenotype. Secretion of mucins was enhanced in BD110-expressing cells, but not in MKK6DA-expressing cells, suggesting that secretion of mucins is independent of the MKK6-p38 MAP kinase cascade. Thus, there may be at least two pathways, p38 MAP kinase-dependent and -independent, which are involved in regulation of cell-cell contact and the protein secretion system, respectively.     

Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis

In this study we sought to clarify the role of the proapoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential deltapsim, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated 'type II' pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.     

From receptors to stress-activated MAP kinases

The cell signaling pathways that culminate in activation of a family of stress-activated MAP kinases are beginning to be defined. Determination of cell life and cell death is known to largely depend on the balance of intrinsic life and death signals within cells. Recently, two representative mammalian stress-activated kinases, the JNK and p38 MAP kinases, have been implicated in determination of cell fate by modifying the life, death and differentiation signals. However, the molecular mechanisms by which extracellular signals are transmitted from membrane receptors to the most upstream kinases in the JNK and p38 signaling modules are not fully understood. This review will provide an overview of current knowledge of molecular links between inflammatory cytokine receptors and stress-activated MAP kinase cascades.     

Cryptotanshinone activates p38/JNK and inhibits Erk1/2 leading to caspase-independent cell death in tumor cells

Cryptotanshinone (CPT), a natural compound isolated from the plant Salvia miltiorrhiza Bunge, is a potential anticancer agent. However, the underlying mechanism is not well understood. Here, we show that CPT induced caspase-independent cell death in human tumor cells (Rh30, DU145, and MCF-7). Besides downregulating antiapoptotic protein expression of survivin and Mcl-1, CPT increased phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-jun N-terminal kinase (JNK), and inhibited phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2). Inhibition of p38 with SB202190 or JNK with SP600125 attenuated CPT-induced cell death. Similarly, silencing p38 or c-Jun also in part prevented CPT-induced cell death. In contrast, expression of constitutively active mitogen-activated protein kinase kinase 1 (MKK1) conferred resistance to CPT inhibition of Erk1/2 phosphorylation and induction of cell death. Furthermore, we found that all of these were attributed to CPT induction of reactive oxygen species (ROS). This is evidenced by the findings that CPT induced ROS in a concentration- and time-dependent manner; CPT induction of ROS was inhibited by N-acetyl-L-cysteine (NAC), a ROS scavenger; and NAC attenuated CPT activation of p38/JNK, inhibition of Erk1/2, and induction of cell death. The results suggested that CPT induction of ROS activates p38/JNK and inhibits Erk1/2, leading to caspase-independent cell death in tumor cells.     

The dietary flavonol fisetin enhances the apoptosis-inducing potential of TRAIL in prostate cancer cells

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous agent that induces apoptosis selectively in cancer cells. Soluble or expressed in immune cells, TRAIL plays an important role in the defense against tumour cells. The resistance of cancer cells to TRAIL immune surveillance is implicated in tumour development. Naturally occurring flavonoids can sensitize TRAIL-resistant cancer cells and augment their apoptotic activity. Fisetin, a dietary flavonol has cancer preventive properties. This study was designed to investigate the effect of fisetin on the TRAIL-induced apoptosis potential in prostate cancer cells. Prostate cancer cell lines represent an ideal model for research in chemoprevention. Cytotoxicity was measured by MTT and LDH assays. Apoptosis was detected using Αnnexin?V-FITC by flow cytometry and fluorescence microscopy. Mito-chondrial membrane potential (ΔΨm) was evaluated using DePsipher staining by fluorescence microscopy. Death receptor (TRAIL-R1 and TRAIL-R2) expression was analysed by flow cytometry. Inhibition of NF-κB (p65) activation was confirmed with an ELISA-based TransAM NF-κB kit. Caspase-8 and caspase-3 activities were determined by colorimetric protease assays. Our study demonstrates that fisetin sensitizes the TRAIL-resistant androgen-dependent LNCaP and the androgen-independent DU145 and PC3 prostate cancer cells to TRAIL-induced death. Fisetin augmented TRAIL-mediated cytotoxicity and apoptosis in prostate cancer LNCaP cells by engaging the extrinsic (receptor-mediated) and intrinsic (mitochondrial) apoptotic pathways. Fisetin increased the expression of TRAIL-R1 and decreased the activity of NF-κB. Co-treatment of cancer cells with TRAIL and fisetin caused significant activation of caspase-8 and caspase-3 and disruption of ΔΨm. Our data indicate the usefulness of fisetin in prostate cancer chemoprevention through enhancement of TRAIL-mediated apoptosis.     

Adenoviral vector expressing CYLD augments antitumor activity of TRAIL by suppression of NF-kappaB survival signaling in hepatocellular carcinoma

CYLD is a tumor suppressor gene related to cylindroma and is negative regulator of NF-kappaB. However, antitumor effect of CYLD has not been reported. The activation of NF-kappaB induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) renders hepatocellular carcinoma (HCC) resistant to TRAIL-mediated cell apoptosis. Here we described that the adenoviral vector expressing CYLD (Ad/hTERT-CYLD) augmented the cytotoxicity of TRAIL in HCC cells by negatively regulating NF-kappaB activity since CYLD could reverse the ubiquitination of TNF receptor-associated factor 2 (TRAF2) and interact with the IkappaB kinasegamma (IKKgamma). The combined treatment of Ad/hTERT-CYLD and a conditionally replicating adenovirus carrying TRAIL gene (ZD55-TRAIL) induced rapid and potent apoptosis in HCC cells, characterized by activation of caspase-3, caspase-8, PARP and the reduction of X-linked inhibitor of apoptosis protein (XIAP). In animal study, the combined treatment could eradicate the BEL7404 xenograft tumors. In contrast, treatment with Ad/hTERT-CYLD or ZD55-TRAIL alone achieved less antitumor effect. In conclusion: CYLD inhibits TRAIL-mediated NF-kappaB activation and enhances the sensitivity of HCC cells to TRAIL-triggered apoptosis. The combined delivery of Ad/hTERT-CYLD and ZD55-TRAIL may be a new useful strategy for HCC or other tumor cells with enhanced NF-kappaB activity.     

Suppression of cFLIP is sufficient to sensitize human melanoma cells to TRAIL- and CD95L-mediated apoptosis

Death ligands such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and certain forms of CD95L are attractive therapeutic options for metastatic melanoma. Since knowledge about the regulation of death receptor sensitivity in melanoma is sparse, we have analysed these signaling pathways in detail. The loss of CD95 or TRAIL-R1, but not of TRAIL-R2, surface expression correlated with apoptosis sensitivity in a panel of melanoma cell lines. In contrast, the expression of proteins of the apical apoptosis signaling cascade (FADD, initiator caspases-8 and cFLIP) did not predict apoptosis sensitivity. Since both TRAIL-R1 and -R2 transmit apoptotic signals, we asked whether cFLIP, highly expressed in several of the cell lines tested, is sufficient to maintain resistance to TRAIL-R2-mediated apoptosis. Downregulation of cFLIP in TRAIL-R2-positive, TRAIL-resistant IGR cells dramatically increased TRAIL sensitivity. Conversely ectopic expression of cFLIP in TRAIL-sensitive, TRAIL-R2-expressing RPM-EP melanoma cells inhibited TRAIL- and CD95L-mediated cell death. Thus, modulation of cFLIP is sufficient to sensitize TRAIL-R2-expressing cells for TRAIL. Taken together, albeit expressing all proteins necessary for death receptor-mediated apoptosis, TRAIL-R1 negative melanoma cells cannot undergo TRAIL- or CD95L-induced apoptosis due to expression of cFLIP. Hence, cFLIP represents an attractive therapeutic target for melanoma treatment, especially in combination with TRAIL receptor agonists.     

Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) sensitizes LNCaP prostate cancer cells to TRAIL-induced apoptosis

Naturally occurring phenolic compounds have been shown to sensitize prostate cancer cells to tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. TRAIL is a potent stimulator of apoptosis in cancer cells and an important immune effector molecule in the surveillance and elimination of developing tumours. However, many cancer cells are resistant to TRAIL-mediated death. In this study, we aimed to determine the mechanisms by which TRAIL resistance can be overcome in prostate cancer cells by 3,5-diprenyl-4-hydroxycinnamic acid (artepillin?C). Artepillin?C is a bioactive component of Brazilian green propolis that possesses antitumour and chemopreventive activities. TRAIL-resistant LNCaP prostate cancer cells were treated with TRAIL and artepillin?C. Cytotoxicity was measured by MTT and lactate dehydrogenase (LDH) assays. Apoptosis was detected using Annexin V-FITC staining by flow cytometry and fluorescence microscopy. Death receptor (DR) (TRAIL-R1/DR4 and TRAIL-R2/DR5) expression was analyzed using flow cytometry. Mitochondrial membrane potential (?ψm) was evaluated using DePsipher staining by fluorescence micro-scopy. The inhibition of NF-κB (p65) activation was confirmed with the ELISA-based TransAM NF-κB kit. Caspase-8 and caspase-3 activities were determined by colorimetric protease assays. The results showed that artepillin?C sensitized the TRAIL-resistant LNCaP cells by engaging the extrinsic (receptor-mediated) and intrinsic (mitochondrial) apoptotic pathways. Artepillin?C increased the expression of TRAIL-R2 and decreased the activity of NF-κB. Co-treatment with TRAIL and artepillin?C induced the significant activation of caspase-8 and caspase-3, as well as the disruption of ?ψm. These findings show that prostate cancer cells can be sensitized to TRAIL-mediated immunoprevention by artepillin?C and confirm the role of phenolic compounds in prostate cancer immunochemoprevention.     

Synergistic induction of tumor cell apoptosis by death receptor antibody and chemotherapy agent through JNK/p38 and mitochondrial death pathway

Using two agonistic monoclonal antibodies specific for each death receptor of TRAIL, 2E12 (anti-human DR4) and TRA-8 (anti-human DR5), we examined the signal transduction of the death receptors in combination with or without chemotherapy agents such as Adriamycin (doxorubicin hydrochloride) and Cisplatin. Our results demonstrated that chemotherapy agents were able to enhance apoptosis-inducing activity of these antibodies against several different types of tumor cell lines through enhanced caspase activation. The combination of the antibodies and chemotherapy agents led to a synergistical activation of the JNK/p38 MAP kinase, which was mediated by MKK4. The combination also caused an increased release of cytochrome c and Smac/DIABLO from mitochondria in parallel with the profound loss of mitochondrial membrane potential. These results suggest that the enhanced activation of the JNK/p38 kinase and the mitochondrial apoptosis pathways play a crucial role in synergistic induction of the death receptor-mediated apoptosis by chemotherapy agents. Thus, the simultaneous targeting of cell surface death receptors with agonistic antibodies and the intracellular JNK/p38 and the mitochondrial death pathways with chemotherapy agents would enhance the efficacy and selectivity of both agents in cancer therapy.     

Signal transduction in anaplastic large cell lymphoma cells (ALCL) mediated by the tumor necrosis factor receptor CD30

Expression of the cytokine receptor CD30 is a characteristic feature of anaplastic large cell lymphoma (ALCL). Reports regarding CD30-mediated signaling in ALCL cells are highly controversial, especially with respect to the regulation of cell survival. In this study, we stimulated 6 ALCL-derived cell lines with immobilized anti-CD30 antibody. CD30-induced cell death was investigated by Western blot and FACS analysis. CD30-dependent cell proliferation and activation was analyzed by applying the trypan blue exclusion method and a luciferase-based ATP assay. The expression of cell cycle relevant proteins and the activation of mitogen-activated protein (MAP) kinases were also examined. We demonstrated that activation of CD30 did not lead to the cleavage of pro-caspase-3. FACS analysis confirmed that in all examined cells cell death was not mediated by CD30. Cell growth was strongly inhibited in 2 of the 6 cell lines and restrained cell growth was accompanied by expression of the cell cycle inhibitor p21(WAF1/CIP1). Furthermore, stimulation of CD30 led to the activation of the p38 MAP kinase but not of the extracellular signal-regulated kinase (ERK) or the jun N-terminal kinase (JNK). Interestingly, activation of CD30 induced a strong synergistic reduction of cell activity, if the p38 MAP kinase activity was blocked by SB203580. The aim of the study was to elucidate CD30-induced signaling in different ALCL-cells. Our results suggest that CD30-mediated apoptosis is not a common feature in this cell type and that p38 MAP kinase is involved in CD30-mediated singal transduction.     

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.