Expression of TRAIL (Apo2L), DR4 (TRAIL receptor 1), DR5 (TRAIL receptor 2) and TRID (TRAIL receptor 3) genes in multidrug resistant human acute myeloid leukemia cell lines that overexpress MDR 1 (HL60/Tax) or MRP (HL60/AR)

Previously we have reported a differential expression of CD95/CD95L and Bcl-2 family of genes in multidrug resistant tumor cells. TRAIL, a member of the TNF receptor family, induces apoptosis in many tumor cells by binding to DR4 (TRAIL receptor 1) and DR5 (TRAIL receptor 2). In contrast, TRAIL-induced apoptosis is prevented by a decoy receptor (DcR1, TRID or TRAIL receptor 3). In the present study, we compared the expression of TRAIL, DR4, DR5, and TRID between a drug sensitive HL60, a myeloid leukemia cell line, and its multidrug resistant (MDR) sublines that either overexpressed MDR 1 gene (HL60/Tax) or MRP gene (HL60/AR), using RT-PCR. TRAIL mRNA was expressed in HL60 cells but was present in low levels in HL60/AR cells and was completely lacking in HL60/Tax cells. Both DR4 and DR5 were undetectable in HL60/Tax but were present at comparable levels in HL60/AR and drug sensitive HL60 cells. TRID were absent in HL60 and HL60/Tax cells, but was present in low but comparable levels in peripheral blood mononuclear cells and HL60/AR cells. These data suggest that the multidrug resistance in MDR HL60 cell lines, regardless of overexpression of MDR 1 or MRP, may be due to different mechanisms. In HL60/AR cells it appears that MDR may be due to decreased expression of TRAIL and constitutive expression of TRID, whereas in HL60/Tax cells, MDR could be due to the absence of TRAIL and/or DR4 and DR5.     

Sensitivity of fresh isolates of soft tissue sarcoma, osteosarcoma and giant cell tumour cells to Apo2L/TRAIL and doxorubicin

Chemotherapy is an established treatment modality for bone sarcomas such as osteosarcoma (OS). However, the use of chemotherapy in high-grade soft tissue sarcomas remains controversial, with the most active chemotherapeutic agent, doxorubicin (DOX), reported to have a response rate of, at best only 34% and most studies reporting lower response rates. Apo2L/TRAIL is a member of the tumour necrosis factor (TNF) family of cytokines and induces death of tumour cells, but not normal cells. Its potent apoptotic activity is mediated through cell surface death domain-containing receptors, DR4/TRAIL-R1 and DR5/TRAIL-R2. We investigated the efficacy of Apo2L/TRAIL as a single agent, and in combination with clinically relevant chemotherapeutic drugs, in fresh isolates of primary malignant cells obtained from biopsy material. The data presented here demonstrate that, in a range of primary bone related tumours, as well as soft tissue sarcomas, chemotherapeutic agents were only moderately effective, in terms of induction of cell death. Apo2L/TRAIL alone had little or no effect on any bone-related tumour or sarcoma in culture. In contrast, the combination of Apo2L/TRAIL and chemotherapeutic drugs produced a significant increase in tumour cell death, with DOX and Apo2L/TRAIL proving to be the most effective combination. These data suggest the potential for Apo2L/TRAIL to increase the effectiveness of chemotherapeutic drugs in bone and soft tissue sarcomas, while perhaps concurrently allowing a reduction in the exposure to drugs such as DOX, and a consequent reduction in toxicity. The synergistic action between these two different classes of agents has yet to be tested in vivo but may prove clinically relevant in the treatment of this refractive class of malignancies.     

Anticancer agents sensitize osteosarcoma cells to TNF-related apoptosis-inducing ligand downmodulating IAP family proteins

Although TNF-related apoptosis-inducing ligand (TRAIL) usually induces cell death in tumor cells, there are some tumor cell types that are resistant to its apoptogenic effects. Some chemotherapeutic drugs, however, can sensitize resistant cancer cells to TRAIL by either upregulating surface TRAIL death receptor expression or by modulating intracellular signalling pathways emanating from TRAIL receptors. U2OS human osteosarcoma cells express TRAIL-R2 but are resistant to TRAIL-induced apoptosis. however, the genotoxic drugs, Doxorubicin and Cisplatin, are able to sensitize U2OS cells to TRAIL, without affecting their surface expression of either death or decoy TRAIL receptors. We demonstrate that Doxorubicin and Cisplatin downmodulate X-IAP, while not affecting FLIP levels in U2OS cells. Selective downmodulation of X-IAP protein synthesis by specific small interference RNA transfection induced a sensitization of U2OS cells to TRAIL comparable to that induced by pharmacological treatment with genotoxic drugs. TRAIL-R2 downmodulation by siRNAs completely abolished the TRAIL-induced apoptosis of genotoxin-treated U2OS cells. Our findings demonstrate that Doxorubicin and Cisplatin do not sensitize U2OS osteosarcoma cells to TRAIL by surface receptor modulation but rather by the removal of the intracellular signalling inhibition generated by X-IAP, suggesting a foreseeable relevant advantage to the therapy of these tumors by the combined regimen of genotoxin-based chemotherapy and TRAIL.     

TRAIL-DISC formation is androgen-dependent in the human prostatic carcinoma cell line LNCaP

We and others have previously described that the androgen-responsive human prostatic carcinoma cell line LNCaP is resistant to TRAIL and that TRAIL-mediated apoptosis in LNCaP is PI3K/Akt-dependent. In this study, we found that LNCaP remained resistant to treatment with TRAIL after androgen deprivation even in the presence of the PI3K/Akt pathway inhibitor wortmannin. This resistance was determined by failure to form the TRAIL-DISC and by decreased TRAIL-R1 and TRAIL-R2 levels after androgen deprivation; the capacity of TRAIL to induce DISC formation was completely restored in the presence of DHT. TRAIL and wortmannin together accelerated processing of caspase-8 on the DISC and apparently the release of caspase-8 from the DISC into the cytoplasm. Surprisingly, we found that wortmannin decreased the total amount of TRAIL-R1, but not TRAIL-R2, in the cells as well as the amount of TRAIL-R1 precipitated by TRAIL. Our data suggest that TRAIL-DISC formation and sensitivity to TRAIL treatment are androgen-dependent in LNCaP.     

Role of the phosphatidylinositol 3'-kinase/PTEN/Akt kinase pathway in tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in non-small cell lung cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)/APO-2L is a member of the TNF superfamily and has been shown to have selective antitumor activity. We here show that TRAIL does not induce apoptosis in some non-small cell lung cancer (NSCLC) cells. These cells are resistant to TRAIL because of the phosphatidylinositol 3'-kinase (PI3-K)-dependent activation of Akt/protein kinase B. The expression of phospho-Akt varies at the functional level but not at the mRNA level in NSCLC cells. Akt induces cell survival in NSCLC cells by blocking the Bid cleavage, upstream of cytochrome c release in the mitochondrial-dependent apoptotic pathway. The use of PI3-K inhibitors, Wortmannin or LY-294002, down-regulates the active Akt and reverses cellular resistance to TRAIL. In addition, genetically altering Akt expression by transfecting dominant negative Akt, sensitizes NSCLC cells to TRAIL. Conversely, transfection of constitutively active Akt into cells that express low, constitutively active Akt, increases TRAIL resistance. Alternate to this approach, transfection with PTEN, a lipid phosphatase, promotes sensitivity to TRAIL, whereas a PTEN mutant (PTEN-G129E) at the catalytic site is inactive in dephosphorylating active Akt. Furthermore, the loss of PTEN activity or overexpression of PI3-K-dependent Akt/protein kinase B activity promotes the survival of NSCLC cells. Modulation of Akt activity by combining pharmacological drugs or genetic alterations of the Akt expression induces cellular responsiveness to TRAIL. Thus, TRAIL can be used to treat NSCLC-resistant cells when combined with agents that down-regulate Akt activity.     

Inducible resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand receptor 2-mediated apoptosis by generation of a blockade at the death domain function

Induction of tumor cell resistance to therapeutics has been a major obstacle in cancer therapy. Targeting of the death receptors by a natural ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), or agonistic monoclonal antibodies against TRAIL receptor 1 (TRAIL-R1) or TRAIL receptor 2 (TRAIL-R2) has been thought to be a promising cancer therapy. To determine whether tumor cells are able to generate a resistance to apoptosis induced by an anti-TRAIL-R2 antibody, TRA-8, we examined the apoptotic response of human breast and ovarian cancer cell lines after treatment with TRA-8. Our results show that tumor cell resistance to TRA-8 can be induced by repeated treatment of tumor cells with low, non-apoptosis-inducing doses of TRA-8. Interestingly, the induced resistance to apoptosis was not due to a global apoptotic defect in tumor cells but rather a selective defect in the TRAIL-R2 signaling pathway. Whereas TRA-8-treated tumor cells developed a selective resistance to TRAIL-R2-mediated apoptosis, the apoptotic responses induced by TRAIL, an anti-TRAIL-R1 antibody (2E12), and other apoptotic stimuli were not impaired. The expression levels of cell surface TRAIL-R2 were not altered and mutations of TRAIL-R2 were not found in the resistant cells. The induced TRA-8 resistance was due to a selective blockade at the level of the death domain and could be reversed by a wide array of chemotherapeutic agents. Proteomic analysis of death-inducing signaling complex formation during TRA-8 treatment shows that the translocation of TRAIL-R2-associated apoptotic proteins was significantly altered. Our results suggest that the prevention of tumor cell resistance to therapeutic agents that target the death receptors must be taken into consideration.     

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.     

Reverse-phase protein microarrays (RPPA) as a diagnostic and therapeutic guide in multidrug resistant leukemia

Reverse-phase microarray assays using phospho-specific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. In the current study, lysates from parental and multidrug resistant (MDR) CEM leukemia cells were spotted onto reverse-phase protein microarrays and probed with a panel of phospho-antibodies to ERK, PCK and Akt pathways. In particular, the Akt pathway is considered to play significant roles in leukemia and Akt inhibitor therapy has been proposed as a potential tool in the treatment of this disease. The RPPA data prompted us to investigate deeper this pathway. Here, we found that whereas total Akt1 protein level is higher in parental CEM cells, the activated isoform content, p-Akt1, increases in doxorubicin-selected CEM cells (MDR-CEM). This was backed up by Western blot analysis, confirming that Akt1 activity/phosphorylation may be up-regulated in MDR-CEM cells. Further exploration of inhibitory therapy in this system was evaluated. The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumor cells. Herein, we describe that in MDR-CEM cells TRAIL responsiveness correlates with a reduced expression of endogenous Akt1, suggesting that the MDR phenotype associated to P-gp sensitizes cells to TRAIL therapy.     

Dual role of ERK2/NF-κB signaling in TRAIL sensitivity

Targeting tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling is a promising approach in cancer treatment. Although ERK and/or NF-κB signaling is involved in the expression of TRAIL receptors (TRAIL-R), the exact underlying mechanisms remain unknown. In this study, we evaluated the role of ERK2 and NF-κB in the cytotoxicity of TRAIL during cisplatin treatment. Cisplatin treatment of neuroepithelioma cells (SK-N-MC) significantly induced ERK2 activation and increased TRAIL cytotoxicity via the upregulation of death receptor 5 (DR5) expression. In partial ERK2 knockdown cell lines that maintained only basal levels of ERK2 activity, cisplatin treatment did not increase ERK2 activity or DR5 expression. These findings indicate that induced (rather than basal) ERK2 activity enhances TRAIL susceptibility via DR5 expression. In complete ERK2 knockdown cell lines with no basal ERK2 activity, DR4, DR5, and DcRs expression levels were increased, and additional treatment with cisplatin did not further increase TRAIL-R expression. Chemical inhibition of ERK2 also enhanced TRAIL cytotoxicity by upregulating DR4 and DR5 expression. These findings indicate that basal ERK2 activity suppresses TRAIL-R expression. Both basal and inducible ERK2 activities regulate TRAIL-R expression via the NF-κB signaling pathway. Overall, our findings suggest that the ERK2/NF-κB signaling pathway has a dual role in TRAIL susceptibility by differentially regulating TRAIL-R expression in the same cellular system.     

Progressive resistance of BTK-143 osteosarcoma cells to Apo2L/TRAIL-induced apoptosis is mediated by acquisition of DcR2/TRAIL-R4 expression: resensitisation with chemotherapy

Apo2 ligand (Apo2L, also known as TRAIL) is a member of the tumour necrosis factor (TNF) family of cytokines that selectively induces the death of cancer cells, but not of normal cells. We observed that recombinant Apo2L/TRAIL was proapoptotic in early-passage BTK-143 osteogenic sarcoma cells, inducing 80% cell death during a 24 h treatment period. Apo2L/TRAIL-induced apoptosis was blocked by caspase inhibition. With increasing passage in culture, BTK-143 cells became progressively resistant to the apoptotic effects of Apo2L/TRAIL. RNA and flow cytometric analysis demonstrated that resistance to Apo2L/TRAIL was paralleled by progressive acquisition of the decoy receptor, DcR2. Blocking of DcR2 function with a specific anti-DcR2 antibody restored sensitivity to Apo2L/TRAIL in a dose-dependent manner. Importantly, treatment of resistant cells with the chemotherapeutic agents doxorubicin, cisplatin and etoposide reversed the resistance to Apo2L/TRAIL, which was associated with drug-induced upregulation of mRNA encoding the death receptors DR4 and DR5. BTK-143 cells thus represent a useful model system to investigate both the mechanisms of acquisition of resistance of tumour cells to Apo2L/TRAIL and the use of conventional drugs and novel agents to overcome resistance to Apo2L/TRAIL.     

RAIDD expression is impaired in multidrug resistant osteosarcoma cell lines

Purpose  To identify the apoptosis genes involved in the multidrug resistant phenotype of osteosarcoma. Methods  Multidrug resistant human osteosarcoma cell line (U-2 OS MR) and a drug sensitive parental cell line (U-2 OS) were both treated with paclitaxel and analyzed by the gene array containing 96 apoptosis associated genes. The different expression of the special apoptosis associated genes were further analyzed by Western blot in the multidrug resistant osteosarcoma cell lines (U-2 OS MR, KH OS R2) and the drug sensitive parental cell lines (U-2 OS, KH OS). One of the disregulated gene, RAIDD, was transfected into the multidrug resistant osteosarcoma cells for functional studies. Results  RAIDD showed signs of significant expression in the U-2 OS cells after being treated with paclitaxel (P < 0.01). However, the induction of RAIDD did not occur in U-2 OS MR cells (P = 0.2). Subsequent analysis by Western blot confirmed the deficiency of the expression of RAIDD protein in U-2 OS MR. On the contrary, the expression of RAIDD could be significantly induced by paclitaxel and doxorubicin in U-2 OS cells as both time and dosage were deciding factors. It also demonstrated the cleavage of PARP associated with RAIDD expression in U-2 OS cells, but not however in U-2 OS MR cells after being treated with paclitaxel or doxorubicin. Similar results were found in osteosarcoma multidrug resistant cell line KH OS R2 and the drug sensitive parental cell line KH OS. Furthermore, over-expression of RAIDD in multidrug resistant cell lines could possibly reverse drug resistant phenotypes. Conclusion  This study indicate that impaired expression of RAIDD in drug induced apoptosis may play a role in the multidrug resistance of osteosarcoma cells.     

Retention of activity by selected anthracyclines in a multidrug resistant human large cell lung carcinoma line without P-glycoprotein hyperexpression.

A subline (COR-L23/R) of the human large cell lung line [corrected] COR-L23, derived by in vivo exposure to doxorubicin, exhibits an unusual multidrug resistant (MDR) phenotype. This subline shows cross-resistance to daunorubicin, vincristine, colchicine and etoposide but does not express P-glycoprotein. Interestingly, COR-L23/R [corrected] shows little or no resistance to a range of structurally-modified analogues of doxorubicin comprising 9-alkyl and/or sugar modified anthracyclines. We have previously identified these same compounds as effective agents against P-glycoprotein-positive MDR cell lines. In contrast to typical MDR cell lines, COR-L23/R [corrected] shows only minimal chemosensitisation by verapamil and no collateral sensitivity to verapamil. Compared to the parental cell line, COR-L23/R [corrected] displays reduced accumulation of doxorubicin and daunorubicin. Accumulation defects were apparent only after 0.5-1 h of incubation of cells with these agents. The rate of daunorubicin efflux was shown to be enhanced by COR-L23/R [corrected] and this efflux was demonstrated to be energy-dependent. The use of anthracyclines which retain activity in MDR cells thus appears to be a valid approach for the circumvention of MDR, not only in cells which express P-glycoprotein, but also where defective drug accumulation is due to other mechanisms possibly involving an alternative multidrug transporter.     

Doxorubicin potentiates TRAIL cytotoxicity and apoptosis and can overcome TRAIL-resistance in rhabdomyosarcoma cells

Doxorubicin (DOX) and ifosfamide (IFO) are the most active single agents in soft tissue sarcomas (STS). Tumour necrosis factor-alpha (TNF-alpha) is used for STS in the setting of isolated limb perfusions. Like TNF-alpha, TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis. In contrast to TNF-alpha preliminary studies suggest that TRAIL lacks systemic side effects. The effects of TRAIL alone and in combination with DOX or 4-hydroxy-IFO were evaluated in the TNF-alpha sensitive rhabdomyosarcoma cell line KYM-1, its 5-fold TNF-alpha sensitive subline KD4 and its >150-fold TNF-alpha resistant subline 37B8R. Membrane expression of TRAIL-receptors DR4 (death receptor 4), DR5 (pro-apoptotic), DcR1 (decoy receptor 1), DcR2 (anti-apoptotic) was assessed by flow cytometry. Cytotoxicity was determined by microculture tetrazolium assays. Apoptosis assays were performed with acridine orange. DOX (doxorubicin) and 4-OH-IFO decreased survival in all cell lines; a 2-fold resistance was observed for both drugs in 37B8R. All cell lines expressed DR4 and DR5, but hardly any DcR1 or DcR2. TRAIL was cytotoxic in KYM-1, even more in KD4 and induced massive apoptosis; 37B8R was >500-fold resistant to TRAIL and little apoptosis could be observed. TRAIL plus DOX showed synergistic cytotoxicity in KYM-1 and 37B8R. TRAIL plus 4-OH-IFO showed addition in all three cell lines. DOX plus TRAIL-induced more cytotoxicity and apoptosis in all cell lines compared to TRAIL alone. In 37B8R, DOX overcame resistance to TRAIL. In KYM-1, KD4 and 37B8R, sensitivity and resistance to TNF-alpha and TRAIL parallels. TRAIL-resistance was independent from expression of TRAIL-receptors. DOX with TRAIL could overcome TRAIL-resistance in 37B8R cells.     

The topoisomerase I inhibitor topotecan increases the sensitivity of prostate tumor cells to TRAIL/Apo-2L-induced apoptosis

PURPOSE.:TRAIL/Apo-2L is cytotoxic against numerous prostate tumor cell lines; however, some lines are more resistant than others. Identification of an agent that increases prostate tumor cell sensitivity to TRAIL/Apo-2L would prove valuable for TRAIL/Apo-2L-mediated tumor therapy. Thus, we examined the effect of combining five clinically approved chemotherapeutic agents with TRAIL/Apo-2L for treating prostate tumor cells. METHODS: Four human prostate tumor cell lines were initially tested for TRAIL/Apo-2L sensitivity. Subsequent studies examined whether the TRAIL/Apo-2L-induced killing of DU-145 cells was augmented in the presence of the chemotherapeutic molecules, as measured by annexin V-FITC/propidium iodide staining. Furthermore, caspase 8 activation and BID cleavage were examined by immunoblotting. RT-PCR and flow cytometry were performed to monitor TRAIL-R1 and TRAIL-R2 levels after chemotherapeutic treatment. RESULTS: DU-145 cells were the least responsive of the prostate tumor cell lines tested to TRAIL/Apo-2L-induced death. Surprisingly, only topotecan, a topoisomerase I inhibitor, when used in combination with rTRAIL/Apo-2L led to significant apoptosis of DU-145 cells, as measured by caspase 8 activation, BID cleavage, and annexin V-FITC/PI staining. Topotecan alone had little to no toxicity on the DU-145 cells. Furthermore, the increase in TRAIL/Apo-2L sensitivity following topotecan treatment correlated with increased expression of TRAIL-R1 and TRAIL-R2 and decreased intracellular levels of the antiapoptotic protein survivin. CONCLUSIONS: Our results define a promising direction for alternative therapies against androgen-independent prostate cancers. The sensitivity of DU-145 cells to TRAIL/Apo-2L was dramatically increased when combined with topotecan, suggesting that low-dose topotecan treatment to upregulate TRAIL-R1 and TRAIL-R2 and downregulate survivin, followed by TRAIL/Apo-2L administration, may be a viable therapy for treating cancer of the prostate.     

Overexpression of LIMK1 promotes migration ability of multidrug-resistant osteosarcoma cells

Multidrug resistance (MDR) to chemotherapy is a major obstacle in the treatment of cancer and the resistance process is multifactorial. Studies on multidrug resistance mechanisms relied on the availability of cancer multidrug resistance cell lines that have been established. In this study we successfully established a multidrug resistance cell line MG63/VCR derived from human osteosarcoma cell line MG63 based on the induction by vincristine. MG63/VCR cells exhibited high resistance to vincristine and other anticancer drugs, accompanied by upregulated expression of MDR-associated genes MDR1, MRP1, and Bcl-2. Notably, we found that MG63/VCR cells exhibited higher migration ability compared to parental MG63 cells. Moreover, we demonstrated that LIMK1, a key regulator of actin cytoskeleton, was overexpressed at both mRNA and protein levels in MG63/VCR cells and the higher LIMK1 protein level was correlated with higher level of phosphorylated cofilin. In addition, knockdown of LIMK1 abolished the higher migration ability of MG63/ VCR cells. These results suggest that LIMK1 overexpression contributes to the invasion and metastasis of drug-resistant osteosarcoma and reveal LIMK as a novel therapeutic target for drug resistant osteosarcoma.     

CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced cytochrome c release.

Death ligands such as CD95 ligand (CD95L) or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) induce apoptosis in radiochemotherapy-resistant human malignant glioma cell lines. The death-signaling TRAIL receptors 2 (TRAIL-R2/death receptor (DR) 5) and TRAIL-R1/DR4 were expressed more abundantly than the non-death-inducing (decoy) receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in 12 human glioma cell lines. Four of the 12 cell lines were TRAIL/Apo2L-sensitive in the absence of a protein synthesis inhibitor, cycloheximide (CHX). Three of the 12 cell lines were still TRAIL/Apo2L-resistant in the presence of CHX. TRAIL-R2 expression predicted sensitivity to apoptosis. Coexposure to TRAIL/Apo2L and cytotoxic drugs such as topotecan, lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, CCNU) or temozolomide resulted in synergistic killing. Synergistic killing was more often observed in cell lines retaining wild-type p53 activity (U87MG, LN-229) than in p53 mutant cell lines (LN-18, T98G, U373MG). Drug exposure resulted in enhanced TRAIL-R2 expression, but decreased TRAIL-R4 expression in U87MG cells. Ectopic expression of dominant-negative p53(V135A) abrogated the drug-induced changes in TRAIL-R2 and TRAIL-R4 expression, but had no effect on synergy. Thus, neither wild-type p53 function nor changes in TRAIL receptor expression were required for synergy. In contrast, synergy resulted possibly from drug-induced cytochrome c release from mitochondria, serving as an amplifier of the TRAIL/Apo2L-mediated cascade of caspase activation. These data provide novel insights into the role of the TRAIL/Apo2L system in malignant gliomas and illustrate that TRAIL/Apo2L-based immunochemotherapy may be an effective therapeutic strategy for these lethal neoplasms.     

Cross-talk between signalling pathways and the multidrug resistant protein MDR-1.

The multidrug resistant protein MDR-1 has been associated with the resistance to a wide range of anti-cancer drugs. Taxol is a substrate for this transporter system and is used in the treatment of a wide range of human malignancies including lung, breast and ovarian cancer. We have generated a series of ovarian cell lines resistant to this compound, all of which overexpress MDR-1 through gene amplification. We present novel evidence that a constitutive activation of the ERK1/2 MAP kinase pathway was also observed although the level of active JNK and p38 remained unchanged. Inhibition of the ERK1/2 MAP kinase pathway using UO126 or PD098059 re-sensitised the Taxol resistant cells at least 20-fold. Importantly, when Mdr-1 cDNA was stably expressed in the wild-type cell line to generate a highly Taxol-resistant sub-line, 1847/MDR5, ERK1/2 MAP kinases again became activated. This result demonstrated that the increased activity of the signalling pathway in the Taxol-resistant lines was directly attributable to MDR-1 overexpression and was not due to the effects of Taxol itself. Additionally, we demonstrated that inhibition of the P13K pathway with LY294002 sensitised the MDR-1-expressing 1847/TX0.5 cells and 1847/MDR5 cells at least 10-fold but had no effect in the wild-type cells. This finding suggests a possible role for this pathway, also, in the generation of resistance to Taxol.