To Tumor Necrosis Factor-related Apoptosis-inducing Ligand Sensitivity Research Articles

A galectin‐3 sequence polymorphism confers TRAIL sensitivity to human breast cancer cells

A common polymorphism, rs4644, coding for Pro64 or His64 of the carbohydrate-binding protein galectin-3, influences the susceptibility of galectin-3 to cleavage by matrix metalloproteinases and is associated with breast cancer incidence. Because forced expression of galectin-3 in a galectin-3 null breast cancer cell line confers sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), the authors sought to determine whether the His64/Pro64 polymorphism of galectin-3 affects the sensitivity to TRAIL. Genomic DNA of breast cell lines was analyzed for the single nucleotide polymorphism rs4644, and cytotoxicity was determined with the MTT assay. When a collection of 9 breast cancer cell lines that express galectin-3 was examined for lectin, galactoside-binding, soluble, 3 (LGALS3) genotype and sensitivity to doxorubicin and TRAIL, doxorubicin sensitivity was not found to be related to LGALS3 genotype. In contrast, none of the 5 cell lines that were homozygous for Pro64 galectin-3 were found to be sensitive to TRAIL, but 2 of 2 homozygous His64 cell lines and 1 of 2 heterozygous His64 cell lines were sensitive to TRAIL. Forced expression of galectin-3 of defined genotype in galectin-3 null cells was used to more directly test the effect of the Pro64His mutation on TRAIL sensitivity. High levels of expression of His64 galectin-3 rendered BT549 cells sensitive to TRAIL and resistant to doxorubicin, but cells expressing Pro64 galectin-3 remained resistant to TRAIL and sensitive to doxorubicin. The results of the current study indicate that the naturally occurring Pro64His mutation in galectin-3 increases sensitivity to death receptor-mediated apoptosis. This finding could be relevant to disparities in breast cancer outcomes across population groups, and could guide the design of future clinical trials of TRAIL-based therapies.

Capsaicin sensitizes malignant glioma cells to TRAIL-mediated apoptosis via DR5 upregulation and survivin downregulation

Capsaicin, a pungent ingredient of red chili peppers, has been reported to possess antitumor activities. Here, we show that subtoxic doses of capsaicin effectively sensitize multiple malignant glioma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Although TRAIL alone mediated partial proteolytic processing of procaspase-3 in glioma cells, cotreatment with capsaicin and TRAIL efficiently restored complete activation of caspases. We found that treatment of various gliomas with capsaicin significantly upregulated DR5, a death receptor of TRAIL, and downregulated the caspase inhibitor survivin. The induction of DR5 was mediated by CHOP/GADD153. The reduction in survivin protein level was associated with downregulation of cyclin B and Cdc2 expression, suggesting that inhibition of Cdc2 activity might contribute to capsaicin-induced survivin downregulation. Taken together, these results indicate that the activity of capsaicin toward DR5 and survivin contributes to the amplification of caspase cascades, thereby restoring TRAIL sensitivity in malignant glioma cells. Interestingly, normal astrocytes were resistant to combined treatment with capsaicin and TRAIL. Neither capsaicin-induced DR5 upregulation/survivin downregulation nor the partial processing of procaspase-3 by TRAIL was induced in astrocytes. Thus, a combined regimen using capsaicin and TRAIL may provide a safe and effective strategy for treating malignant gliomas.

A Novel PTEN-Dependent Link to Ubiquitination Controls FLIPS Stability and TRAIL Sensitivity in Glioblastoma Multiforme

Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIP(S), and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIP(S) protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIP(S) polyubiquitination. FLIP(S) half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIP(S) E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIP(S) or with the FLIP(S)-containing death inducing signal complex. Small interfering RNA-mediated suppression of AIP4 levels in PTEN WT cells decreased FLIP(S) ubiquitination, prolonged FLIP(S) half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIP(S) steady-state levels, and suppressed FLIP(S) ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIP(S) ubiquitination, FLIP(S) stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability.

Glycogen synthase kinase-3 inhibitors augment TRAIL-induced apoptotic death in human hepatoma cells

Hepatocellular carcinoma (HCC) displays a striking resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Therefore, the characterization of pharmacological agents that overcome this resistance may provide new therapeutic modalities for HCC. Here, we examined whether glycogen synthase kinase-3 (GSK-3) inhibitors could restore TRAIL sensitivity in hepatoma cells. To this aim, the effects of two GSK-3 inhibitors, lithium and SB-415286, were analyzed on TRAIL apoptotic signaling in human hepatoma cell lines in comparison with normal hepatocytes. We observed that both inhibitors sensitized hepatoma cells, but not normal hepatocytes, to TRAIL-induced apoptosis by enhancing caspase-8 activity and the downstream recruitment of the mitochondrial machinery. GSK-3 inhibitors also stabilized p53 and the down-regulation of p53 by RNA interference abolished the sensitizing effect of lithium on caspase-3 activation. Concomitantly, GSK-3 inhibitors strongly activated c-Jun N-terminal kinases (JNKs). The pharmacological inhibition of JNKs with AS601245 or SP600125 resulted in an earlier and stronger induction of apoptosis indicating that activated JNKs transduced protective signals and provided an anti-apoptotic balance to the pro-apoptotic effects of GSK-3 inhibitors. These findings demonstrate that GSK-3 exerts a negative and complex constraint on TRAIL apoptotic signaling in hepatoma cells, which can be greatly alleviated by GSK-3 inhibitors. Therefore, GSK-3 inhibitors may open new perspectives to enhance the anti-tumor activity of TRAIL in HCC.

Imatinib enhances human melanoma cell susceptibility to TRAIL-induced cell death: relationship to Bcl-2 family and caspase activation

In order to define genetic determinants of primary and metastatic melanoma cell susceptibility to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), we have applied oligonucleotide microarrays to TRAIL-sensitive primary T1 cells and TRAIL-resistant metastatic G1 cells treated or not with TRAIL. T1 and G1 cells are isogenic melanoma cell subclones. We examined 22 000 spots, 4.2% of which displayed differential expression in G1 and T1 cells. Cell susceptibility to TRAIL-mediated apoptosis was found to be correlated with gene expression signatures in this model. Some of the differentially expressed genes were identified as involved in ATP-binding and signaling pathways, based on previously published data. Further analysis provided evidences that c-kit was overexpressed in G1 cells while it was absent in T1 cells. The c-kit inhibitor, imatinib, did not restore TRAIL sensitivity, excluding a role for c-kit in TRAIL resistance in G1 cells. Surprisingly, imatinib inhibited cell proliferation and TRAIL-mediated apoptosis in melanoma cells. We investigated the possible involvement of several molecules, including c-ABL, platelet-derived growth factor receptor (PDGFR), cellular FADD-like interleukin-1 alpha-converting enzyme-like inhibitory protein (c-FLIP)(L/S), Fas-associated DD kinase, p53, p21(WAF1), proteins of B-cell leukemia/lymphoma 2 (Bcl-2) family and cytochrome c. Imatinib did not modulate the expression or activation of its own targets, such as c-ABL, PDGFRalpha and PDGFRbeta, but it did affect the expression of c-FLIP(L), BCL2-associated X protein (Bax) and Bcl-2. Moreover, c-FLIP(L) knockdown sensitized T1 cells to TRAIL-mediated apoptosis, with a sensitivity similar to that of cells previously treated with imatinib. More notably, we found that the resistance to TRAIL in G1 cells was correlated with constitutive c-FLIP(L) recruitment to the DISC and the inhibition of caspase 8, 3 and 9 processing. Moreover, c-FLIP(L) knockdown partly restored TRAIL sensitivity in G1 cells, indicating that the expression level of c-FLIP(L) and its interaction with TRAIL receptor2 play a crucial role in determining TRAIL resistance in metastatic melanoma cells. Our results also show that imatinib enhances TRAIL-induced cell death independently of BH3-interacting domain death agonist translocation, in a process involving the Bax:Bcl-X(L) ratio, Bax:Bcl-X(L)/Bcl-2 translocation, cytochrome c release and caspase activation. Our data indicate that imatinib sensitizes T1 cells by directly downregulating c-FLIP(L), with the use of an alternative pathway for antitumor activity, because PDGFRalpha is not activated in T1 cells and these cells do not express c-kit, c-ABL or PDGFRbeta. Caspase cascade activation and mitochondria also play a key role in the imatinib-mediated sensitization of melanoma cells to the proapoptotic action of TRAIL.

Subtoxic concentration of doxorubicin enhances TRAIL-induced apoptosis in human prostate cancer cell line LNCaP

Most tumor cells are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis but sparing to normal cells, thus providing therapeutic potential for clinical use. Some tumor cells are resistant to TRAIL-induced cell death while the sensitivity could be recruited with the existence of some chemical agents. In this study, human prostatic cancer cell line LNCaP was found to be resistant to TRAIL-induced apoptosis while it could be restored to TRAIL sensitivity with combination treatment of low concentration of doxorubicin. TRAIL receptor-1 (DR4) and TRAIL receptor-2 (DR5) were upregulated under the treatment of doxorubicin and verified to be responsible for TRAIL-mediated signal transduction. Furthermore, caspase-8 and caspase-3 were activated and drove their autocleavage into programmed cell death. Interestingly, apoptosis-inhibitory protein c-FLIP, but not Bcl-2 and XIAP was downregulated after doxorubicin treatment. Taken together, these findings suggested that the pathway of cell apoptosis induced by TRAIL was intact but under negative control. Subtoxic concentration of doxorubicin effectively boosted TRAIL sensitivity via depletion of antiapoptotic protein. These findings support the new strategies for killing tumors with TRAIL and chemical agents.

Sensitization of human glioblastomas to tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) by NF‐κB inhibitors

Glioblastoma is the most malignant form of primary brain tumor in adults, with no effective therapy and a low survival rate. TRAIL is a member of the TNF family, which selectively induces apoptosis in certain neoplastic cells, but not normal cells. In this study, we investigated the sensitivity of 7 human glioblastoma cell lines to TRAIL and the expression in them of TRAIL receptors. TRAIL exhibited significant cytotoxicity in 5 of 7 glioma cell lines. These glioblastoma cell lines expressed TRAIL-R2, but not TRAIL-R1, R3, or R4. However, no correlation was observed between the TRAIL sensitivity and the TRAIL-R2 expression level, suggesting that there is an additional determinant of TRAIL sensitivity. Treatments with NF-kappaB inhibitors, such as LLnL, MG132, and SN50, significantly increased the sensitivity of glioma cells to TRAIL. These results suggested that activation of NF-kappaB is a protective mechanism against TRAIL-induced cell death in some glioma cells, and thus NF-kappaB inhibitors may be useful to improve the clinical treatment of glioblastoma with TRAIL.

Death receptor 4 (DR4) efficiently kills breast cancer cells irrespective of their sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Breast cancer cells are generally resistant to induction of apoptosis by treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we demonstrate that both TRAIL-sensitive and TRAIL-resistant breast cancer cell lines can be efficiently killed by overexpression of the TRAIL receptor, death receptor 4 (DR4). The extent of cell death depended on the strength of the promoter driving DR4 expression. When driven by the strong CMV promoter, expression of DR4 killed over 90% of cells in five out of six cell lines tested in the absence of exogenous TRAIL. When driven by the relatively weak tumor-specific hTERT promoter, DR4 was less effective alone, but sensitized cells to killing by TRAIL. The extent of TRAIL sensitization depended on the magnitude of hTERT promoter activity. MCF-7 cells were relatively resistant to the action of DR4. We compared expression of the genes involved in transduction and execution of the death receptor-initiated apoptotic stimuli between MCF-7 and DR4-sensitive cell lines. We confirmed that in the panel of cell lines, MCF-7 was the only line deficient in expression of caspase 3. Bcl-2 and FLIP proteins, implicated in suppression of TRAIL-induced apoptosis, were expressed at a higher level.