Itch/AIP4-independent proteasomal degradation of cFLIP induced by the histone deacetylase inhibitor SAHA sensitizes breast tumour cells to TRAIL

The histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) is undergoing clinical trials as an antitumor drug and has received regulatory approval for cancer treatment. Here, we show that pre-treatment of human breast cancer cells with SAHA makes them susceptible to apoptosis induced by TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). The apoptosis of breast tumour cells induced by TRAIL is blocked at the level of apical activation of caspase-8 and SAHA enhances the TRAIL-induced processing of procaspase-8. Consequently, a TRAIL associated pathway of apoptosis operated via mitochondria is activated in cells treated with SAHA. Interestingly, degradation of cellular FLICE-inhibitory proteins (cFLIP_L and cFLIP_S) by an ubiquitin/proteasome-dependent Itch/AIP4-independent mechanism is observed upon exposure to SAHA. Targeting cFLIP_L directly with siRNA oligonucleotides also sensitizes human breast tumour cells to TRAIL-induced apoptosis. Furthermore, cFLIP_L over-expression significantly inhibits the apoptosis elicited through the combined effects of SAHA and TRAIL. Together, these results indicate that SAHA sensitizes breast cancer cells to TRAIL-induced apoptosis by facilitating the activation of early events in the apoptotic TRAIL pathway. Therefore, the combination of TRAIL and SAHA may represent a therapeutic tool to combat breast tumours.     

P-glycoprotein enhances TRAIL-triggered apoptosis in multidrug resistant cancer cells by interacting with the death receptor DR5

The death-inducing cytokine TRAIL is a promising agent for anticancer therapy since it preferentially kills cancer versus normal cells; however, some cancer cells are TRAIL-resistant. We initially explored whether overexpression of the MDR1 gene product P-glycoprotein (P-gp), which causes multidrug resistance (MDR) in cancer cells, also contributes to TRAIL-resistance. Surprisingly, our results revealed that P-gp-overexpression enhances TRAIL-induced apoptosis not only in neoplastic cells transfected with the MDR1 gene but also in MDR variants selected with cytotoxic anticancer agents. Mechanistic analysis of TRAIL-induced apoptosis in the MDR1-transfected MCF-7 breast cancer cell line BC-19 revealed that TRAIL-triggered significantly more apoptosis in these cells compared with parental MCF-7 cells by binding to the TRAIL receptor DR5. DR5 but not DR4 engagement by TRAIL attenuated cellular ATP levels by robustly stimulating P-gp ATPase activity, and thus triggered P-gp-dependent apoptosis by depletion of the cellular ATP pool. In addition to hyperactive P-gp-mediated ATP hydrolysis, TRAIL-induced, P-gp-potentiated apoptosis was associated with activation of caspases-6, -7, -8, and -9; Bid cleavage; and mitochondrial depolarization. P-gp interacted with the TRAIL receptors DR4, DR5, and DcR1 in plasma membranes and enhanced TRAIL binding to DR5. Interestingly, the decreased level of the decoy TRAIL receptor, DcR1, in BC-19 cells further sensitized these cells to TRAIL. Therefore, both extrinsic and intrinsic apoptosis pathways are involved in this process. These findings for the first time reveal that TRAIL treatment preferentially causes apoptosis in P-gp-overexpressing MDR cells, and suggests significant clinical implications for the use of TRAIL in treating neoplasms that have failed chemotherapy.     

Trichostatin A sensitizes human ovarian cancer cells to TRAIL-induced apoptosis by down-regulation of c-FLIPL via inhibition of EGFR pathway

TRAIL-resistant cancer cells can be sensitized to TRAIL by combination therapy. In this study, we investigated the effect of trichostatin A (TSA), a histone deacetylase inhibitor, to overcome the TRAIL resistance in human ovarian cancer cells. Co-treatment of human ovarian cancer cells with TSA and TRAIL synergistically inhibited cell proliferation and induced apoptosis. The combined treatment of ovarian cancer SKOV3 cells with TSA and TRAIL significantly activated caspase-8 and truncated Bid, resulting in the cytosolic accumulation of cytochrome c as well as the activation of caspase-9 and -3. Moreover, we found that down-regulation of c-FLIP_L might contribute to TSA-mediated sensitization to TRAIL-induced apoptosis in SKOV3 cells, and this result was supported by showing that down- or up-regulation of c-FLIP_L with transfection of siRNA or plasmid sensitized or made SKOV3 cells resistant to TRAIL-induced apoptosis, respectively. TSA or co-treatment with TSA alone and TRAIL also resulted in down-regulation of EGFR1/2 and dephosphorylation of its downstream targets, AKT and ERK. Treatment of SKOV3 cells with PKI-166 (EGFR1/2 inhibitor), LY294002 (AKT inhibitor), and PD98059 (ERK inhibitor) decreased c-FLIP_L expression and co-treatment with TRAIL further reduced the level of c-FLIP_L, respectively, as did TSA. Collectively, our data suggest that TSA-mediated sensitization of ovarian cancer cells to TRAIL is closely correlated with down-regulation of c-FLIP_L via inhibition of EGFR pathway, involving caspase-dependent mitochondrial apoptosis, and combination of TSA and TRAIL may be an effective strategy for treating TRAIL-resistant human ovarian cancer cells.     

Verrucarin A sensitizes TRAIL-induced apoptosis via the upregulation of DR5 in an eIF2α/CHOP-dependent manner

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is one of the most promising candidates for new cancer therapeutics. However, resistance to TRAIL in some cancers remains a current problem in recent. The protein-folding compartment of the endoplasmic reticulum (ER) is particularly sensitive to disturbances, which, if severe, may trigger apoptosis. Therefore, we examined whether verrucarin A (VA) sensitize TRAIL-induced apoptosis in cancer cells by induction of ER stress. We first found that VA induces a major molecule of ER stress, CCAAT/enhancer binding protein homologous protein (CHOP)-dependent DR5 induction and subsequently increases TRAIL-induced cleavage of caspases and PARP in TRAIL-resistant Hep3B cells. Importantly, the transient knockdown using siRNA for CHOP abrogated VA-induced DR5 expression and attenuated TRAIL-induced apoptosis. Treatment with VA also increased the levels of phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), which is a common cellular response of ER stress. Furthermore, salubrinal, a specific eIF2α phosphorylation-inducing agent, increased CHOP and DR5 expression in the presence of VA. In contrast, transfection of mutant-eIF2α significantly reversed VA-induced apoptosis with downregulation of CHOP-dependent DR5 expression. Therefore, VA-induced eIF2α phosphorylation seemed to be important for CHOP and DR5 upregulation and TRAIL-induced apoptosis. In addition, generation of reactive oxygen species (ROS) is an effector molecular in sensitization of VA-induced ER stress. We concluded that VA triggers TRAIL-induced apoptosis by eIF2α/CHOP-dependent DR5 induction via ROS generation.     

Targeting androgen receptor and trail:a novel treatment paradigm for breast cancer

OBJECTIVE TNF-related apoptosis-inducing ligand(TRAIL)is a promising cancer therapeutic agent due to its minimal toxicity to normal tissues and remarkable apoptotic activity in tumors.However,most breast cancer cells are resistant to TRAIL-induced apoptosis.Our objectives are to investigate the underlying molecular mechanisms and to develop strategies to overcome such resistance.METHODS To identify modulators of TRAIL-induced apoptosis,we carried out a genome wide si RNA screen.To validate the screening result,we either silenced or overexpressed the identified genes in various breast cancer cells and changes in growth and TRAIL-induced cell apoptosis were determined in vitro and in an orthotopic xenograft mouse model.Finally,we investigated whether small molecules targeting the identified genes improve the effectiveness of TRAIL-therapy.RESULTS We unexpectedly identified androgen receptor(AR)to be responsible for TRAIL resistance.While AR is classically viewed as the key factor in prostate cancer progression,we found that AR expression levels were markedly elevated in human invasive breast cancer specimens including triple-negative breast cancers(TNBC)that are highly aggressive with poor prognosis.Importantly,breast cancer cell lines express different levels of AR that correlated with their TRAIL resistance.AR overexpression in MDA-MB-231 and MDA-MB-436 cells suppressed the TRAIL sensitivity whereas knockdown of AR rendered MCF-7 and MDA-MB-453 cells sensitive to TRAIL-induced apoptosis.AR overexpression also induced TRAIL resistance in breast tumors in vivo.Further,we observed an upregulation of the TRAIL receptor,death receptor 5(DR5)in breast cancer cells,following the removal or inhibition of AR by its antagonists Casodex and MDV3100.Treatment with AR antagonists also enhanced TRAIL-induced breast cancer cell apoptosis.CONCLUSION AR signaling suppresses TRAIL-induced breast cancer cell apoptosis,in part,by suppressing DR5 expression,and a combination of AR antagonists together with TRAIL may be a novel and effective therapy for TNBC.     

Cooperation between Apo2L/TRAIL and bortezomib in multiple myeloma apoptosis

The proteasome inhibitor bortezomib is currently an important drug for treatment of relapsed and refractory multiple myeloma (MM) and for elderly patients. However, cells from some patients show resistance to bortezomib. We have evaluated the possibility of improving bortezomib therapy with Apo2L/TRAIL, a death ligand that induces apoptosis in MM but not in normal cells. Results indicate that cotreatment with low doses of bortezomib significantly increased apoptosis of MM cells showing partial sensitivity to Apo2L/TRAIL. Bortezomib treatment did not significantly alter plasma membrane amount of DR4 and DR5 but increased Apo2L/TRAIL-induced caspase-8 and caspase-3 activation. Apo2L/TRAIL reverted bortezomib-induced up-regulation of β-catenin, Mcl-1 and FLIP, associated with the enhanced cytotoxicity of combined treatment. More important, some cell lines displaying resistance to bortezomib were sensitive to Apo2L/TRAIL-induced apoptosis. A cell line made resistant by continuous culture of RPMI 8226 cells in the presence of bortezomib (8226/7B) was highly sensitive to Apo2L/TRAIL-induced apoptosis. Moreover, RPMI 8226 cells overexpressing Mcl-1 (8226/Mcl-1) or Bcl-x_L (8226/Bcl-x_L) also showed enhanced resistance to bortezomib, but co-treatment with Apo2L/TRAIL reverted this resistance. These results indicate that Apo2L/TRAIL can cooperate with bortezomib to induce apoptosis in myeloma cells and can be an useful adjunct for MM therapy.     

The antitumor lignan Nortrachelogenin sensitizes prostate cancer cells to TRAIL-induced cell death by inhibition of the Akt pathway and growth factor signaling

Prostate cancer cells frequently develop resistance toward androgen-deprivation and chemotherapy. To identify new approaches to treat androgen-dependent prostate cancer, we have performed a structure–activity analysis of lignan polyphenols for cancer cell specific sensitization to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand that has ability to induce tumor-specific cell death. In this study, we report that the lignan nortrachelogenin (NTG) is the most efficient of the 27 tested lignan compounds in sensitizing prostate cancer cells to TRAIL-induced apoptosis. Importantly, pretreatment with NTG does not sensitize a non-malignant prostate cell line to TRAIL-induced cell death. The structural comparison of lignans reveals that the dibenzylbutyrolactone skeleton is required for the apoptosis-sensitizing activity, while substitutions at the aromatic rings do not seem to play a critical role in this lignan function. Our study also characterizes the cellular effects and molecular mechanisms involved in NTG anticancer activity. We previously reported that specific lignans inhibit the Akt survival-signaling pathway in concert with TRAIL sensitization. While NTG is also shown to be a effective inhibitor of Akt signaling, in this study we further demonstrate that NTG potently inhibits tyrosine kinase (RTK) activation in response to growth factors, such as insulin and insulin-like growth factor I (IGF-I). Our results identify NTG as a novel agent for prostate cancer therapy with ability to inhibit Akt membrane localization and activity as well as the activation of growth factor receptors (GFRs), thereby efficiently synergizing with TRAIL exposure.     

Sorafenib induces apoptotic cell death in human non-small cell lung cancer cells by down-regulating mammalian target of rapamycin (mTOR)-dependent survivin expression

Sorafenib, a multikinase inhibitor, is emerging as a promising targeted agent that may possess antitumor activity against a broad range of cancers. The mechanism by which sorafenib induces lung cancer cell death and apoptosis, however, is not understood. In the present study, we provide evidence that sorafenib acts through inhibition of mammalian target of rapamycin (mTOR) to down-regulate survivin and promote apoptotic cell death in human non-small cell lung cancer (NSCLC) cells. Sorafenib induced ATF4-mediated Redd1 expression, leading to mTOR inhibition—the upstream signal for down-regulation of survivin. Overexpression of survivin reduced sorafenib-induced apoptosis, whereas silencing survivin using small interfering RNA (siRNA) enhanced it, supporting the interpretation that down-regulation of survivin is involved in sorafenib-induced cell death in human NSCLC cells. Furthermore, sorafenib abolished the induction of survivin that normally accompanies IGF-1-stimulated mTOR activation. We further found that Redd1-induced mTOR down-regulation and ATF4/CHOP-induced expression of the TRAIL receptor DR5 associated with sorafenib treatment helped sensitize cells to TRAIL-induced apoptosis. Our study suggests that sorafenib mediates apoptotic cell death in human NSCLC cells through Redd1-induced inhibition of mTOR and subsequent down-regulation of survivin, events that are associated with sensitization to TRAIL-induced apoptotic cell death.     

Cardamonin sensitizes tumour cells to TRAIL through ROS- and CHOP-mediated up-regulation of death receptors and down-regulation of survival proteins

BACKGROUND AND PURPOSE

TNF-related apoptosis-inducing ligand (TRAIL) is currently in clinical trials as a treatment for cancer, but development of resistance is a major drawback. Thus agents that can overcome resistance to TRAIL are urgently needed. Cardamonin (2′,4′-dihydroxy-6′-methoxychalcone) has been shown to affect cell growth by modulating various cell signalling pathways. Hence, we investigated the effect of cardamonin on the actions of TRAIL.

EXPERIMENTAL APPROACH

The effect of cardamonin on TRAIL was measured by plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3 in human colon cancer cells.

KEY RESULTS

Cardamonin potentiated TRAIL-induced apoptosis and this correlated with up-regulation of both the TRAIL death receptor (DR) 4, 5 at mRNA and protein levels. TRAIL-decoy receptor DcR1 was down-regulated by cardamonin. Induction of DRs by cardamonin occurred in a variety of cell types. Gene silencing of the DRs by small interfering RNA (siRNA) abolished the effect of cardamonin on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the DR. Induction of the DR by cardamonin was p53-independent but required CCAAT/enhancer binding protein homologous protein (CHOP); cardamonin induced CHOP, and its silencing by siRNA eliminated the induction of DR5. Cardamonin increased the production of reactive oxygen species (ROS) and quenching ROS abolished its induction of receptors and enhancement of TRAIL-induced apoptosis. Cardamonin also decreased the expression of various cell survival proteins.

CONCLUSIONS AND IMPLICATIONS

Cardamonin potentiates TRAIL-induced apoptosis through ROS-CHOP-mediated up-regulation of DRs, decreased expression of decoy receptor and cell survival proteins. Thus, cardamonin has the potential to make TRAIL more effective as an anticancer therapy.     

蛇床子素联合肿瘤坏死因子诱导凋亡配体对乳腺癌干细胞的杀伤效应

目的 探讨蛇床子素联合肿瘤坏死因子诱导凋亡配体[tumor necrosis factor （TNF）-related apoptosis inducing ligand,TRAIL]对乳腺癌干细胞的杀伤效应及机制。方法 用TRAIL及蛇床子素体外处理MCF-7乳腺癌非干细胞及MCF-7乳腺癌干细胞,MTT法检测乳腺癌细胞的细胞活力;流式细胞术检测乳腺癌细胞的凋亡;Western blot试验检测caspase-9和caspase-3的活化,凋亡酶激活因子（apoptotic protease activating facter-1,Apaf-1）的表达水平,细胞色素C的释放;免疫共沉淀法检测Apaf-1和caspase-9前体的相互作用。结果 MCF-7肿瘤干细胞对TRAIL的敏感性显著低于MCF-7非干细胞。在MCF-7肿瘤干细胞的培养体系中加入蛇床子素能显著提高TRAIL对MCF-7肿瘤干细胞的细胞活力抑制率。Western blot实验结果表明,蛇床子素能显著上调MCF-7肿瘤干细胞中Apaf-1的表达水平,但不影响细胞色素C的释放。在MCF-7肿瘤干细胞中转染Apaf-1 siRNA后,蛇床子素联合TRAIL对MCF-7肿瘤干细胞的协同杀伤活性受到显著抑制。另外,免疫共沉淀实验结果表明,蛇床子素联合TRAIL能显著诱导MCF-7肿瘤干细胞中Apaf-1与caspase-9前体的相互作用,并使之发生活化。结论 蛇床子素通过上调Apaf-1的表达促进TRAIL对乳腺癌干细胞caspase-9的活化,从而增强TRAIL对乳腺癌干细胞的凋亡诱导效应。     

凋亡抑制蛋白在TRAIL诱导胃癌细胞凋亡中的作用

目的探讨凋亡抑制蛋白(inhibitor of apoptosis pro-teins,IAP)在调节胃癌细胞对肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)敏感性方面的作用。方法 PI染色流式细胞术检测细胞凋亡;Western blot检测caspase-3、PARP、XIAP、Survivin、cIAP1和cIAP2的表达。结果 TRAIL能诱导胃癌细胞凋亡,BGC-823细胞较SGC-7901细胞对TRAIL更敏感。caspase-3的活化及PARP的裂解在TRAIL作用早期即出现,且BGC-823细胞较SGC-7901细胞发生得更快。4种IAP蛋白在两株胃癌细胞中都组成性地高表达,Survivin和cIAP1在TRAIL处理前后无变化。而XIAP在BGC-823细胞中明显下降,在SGC-7901细胞中无改变。cIAP2在TRAIL作用后两株细胞中均有所降低。结论 TRAIL诱导胃癌细胞凋亡可能在活化的caspase-3水平受调控,XIAP能保护胃癌细胞免于凋亡。     

Enhanced TRAIL-mediated apoptosis in prostate cancer cells by the bioactive compounds neobavaisoflavone and psoralidin isolated from Psoralea corylifolia

Numerous compounds detected in medical plants and dietary components or supplements possess chemopreventive, antitumor and immunomodulatory properties. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important endogenous anticancer factor that induces apoptosis selectively in cancer cells. However, some tumor cells are resistant to TRAIL-mediated apoptosis. Naturally occurring agents could sensitize TRAIL-resistant cancer cells and augment their apoptotic activity.We examined the cytotoxic and apoptotic effects of neobavaisoflavone and psoralidin in combination with TRAIL on LNCaP prostate cancer cells. The cytotoxicity was evaluated by MTT and LDH assays. The apoptosis was detected using Annexin V-FITC by flow cytometry and fluorescence microscopy. The LNCaP cells were shown to be resistant to TRAIL-induced apoptosis. Our study demonstrated that neobavaisoflavone and psoralidin sensitized TRAIL-resistant cells and markedly augmented TRAIL-mediated apoptosis and cytotoxicity in prostate cancer cells. Cotreatment of LNCaP cells with 100 ng/ml TRAIL and 50 μM neobavaisoflavone or 50 μM psoralidin increased the percentage of the apoptotic cells to 77.5±0.5% or 64.4±0.5%, respectively. The data indicate the potential role of the bioactive compounds isolated from the medicinal plant Psoralea corylifolia (neobavaisoflavone and psoralidin) in prostate cancer chemoprevention through enhancement of TRAIL-mediated apoptosis.     

Acute sensitization of colon cancer cells to inflammatory cytokines by prophase arrest

Understanding how colon cancer cells survive within the inflammatory milieu of a tumor, and developing approaches that increase their sensitivity to inflammatory cytokines, may ultimately lead to novel approaches for colon cancer therapy and prevention. Analysis of a number of chemopreventive and therapeutic agents reveal that HDAC inhibitors are particularly adept at sensitizing colon cancer cells TNF or TRAIL mediated apoptosis. In vivo data are consistent with an interaction between SAHA and TNF in inducing apoptosis, as AOM-induced colon tumors express elevated levels of TNF and are more sensitive to SAHA administration. Cell cycle analysis and time-lapse imaging indicated a close correspondence between SAHA-induced prophase arrest and TNF or TRAIL-induced apoptosis. Prophase arrest induced by the Aurora kinase inhibitor VX680 likewise sensitized cells to TNF and TRAIL, with siRNA analysis pointing to Aurora kinase A (and not Aurora kinase B) as being the relevant target for this sensitization. We propose that agents that promote prophase arrest may help sensitize cancer cells to TNF and other inflammatory cytokines. We also discuss how circumvention of an early mitotic checkpoint may facilitate cancer cell survival in the inflammatory micro-environment of the tumor.     

P-glycoprotein-dependent resistance of cancer cells toward the extrinsic TRAIL apoptosis signaling pathway

The TNF-related apoptosis-inducing ligand (TRAIL or Apo2L) preferentially cause apoptosis of malignant cells in vitro and in vivo without severe toxicity. Therefore, TRAIL or agonist antibodies to the TRAIL DR4 and DR5 receptors are used in cancer therapy. However, many malignant cells are intrinsically resistant or acquire resistance to TRAIL. It has been previously proposed that the multidrug transporter P-glycoprotein (Pgp) might play a role in resistance of cells to intrinsic apoptotic pathways by interfering with components of ceramide metabolism or by modulating the electrochemical gradient across the plasma membrane. In this study we investigated whether Pgp also confers resistance toward extrinsic death ligands of the TNF family. To this end we focused our study on HeLa cells carrying a tetracycline-repressible plasmid system which shuts down Pgp expression in the presence of tetracycline. Our findings demonstrate that expression of Pgp is a significant factor conferring resistance to TRAIL administration, but not to other death ligands such as TNF-α and Fas ligand. Moreover, blocking Pgp transport activity sensitizes the malignant cells toward TRAIL. Therefore, Pgp transport function is required to confer resistance to TRAIL. Although the resistance to TRAIL-induced apoptosis is Pgp specific, TRAIL itself is not a direct substrate of Pgp. Pgp expression has no effect on the level of the TRAIL receptors DR4 and DR5. These findings might have clinical implications since the combination of TRAIL therapy with administration of Pgp modulators might sensitize TRAIL resistant tumors.     

Molecular crosstalk between TRAIL and natural antioxidants in the treatment of cancer

The endogenous protein, tumour necrosis factor receptor apoptosis-inducing ligand (TRAIL), induces apoptosis in a wide variety of transformed and cancer cells but has little or no effect on normal cells. Therefore, TRAIL is considered to be a tumour-selective, apoptosis-inducing cytokine and a promising new candidate for cancer prevention and treatment. Some cancer cells are however resistant to TRAIL-induced apoptosis, but treatment in combination with conventional chemotherapeutic drugs or radiation generally restores TRAIL sensitivity in those cells. A novel class of molecules exhibiting synergy with TRAIL but devoid of major side effects are emerging as alternative approaches to treat resistant cancer cells, including natural antioxidants such as sulphoraphane or the flavonoids curcumin, quercetin, resveratrol, baicalein and wogonin. In this issue of the BJP, Lee et al. demonstrate that treatment of TRAIL-resistant cancer cells with wogonin restores TRAIL-induced cell death in a reactive oxygen species-dependent manner through up-regulation of p53 and Puma.     

Modulation of TRAIL-induced apoptosis by HDAC inhibitors

Triggering apoptosis, the cell's intrinsic death program, is a promising approach for cancer therapy. TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily of death inducing ligands, is of special interest for cancer therapy, since TRAIL has been shown to predominantly kill cancer cells, while sparing normal cells. However, since many cancers fail to undergo apoptosis in response to TRAIL treatment, TRAIL-based combination therapies have been developed for cancer-cell specific sensitization towards TRAIL. Chromatin remodelling plays an important role in gene regulation and aberrant architecture of the chromatin has been implicated in tumor formation and progression. In recent years, HDAC inhibitors (HDACI) that reverse aberrant epigenetic changes have emerged as a potential strategy to sensitize cancer cells for TRAIL-induced apoptosis. Synergistic tumor cell death has been reported in a variety of human cancers using different HDACI together with TRAIL. Here, recent advances in the understanding of the molecular events that underlie the synergistic interaction of HDACI and TRAIL are discussed as well as how this knowledge can be translated into the design of cancer-selective novel therapeutics.     

Impaired activation of caspases and prevention of mitochondrial dysfunction in the metastatic colon carcinoma CC531s-m2 cell line

In a previous paper we described the properties of a rapidly metastasizing cell line CC531s-m2 derived from the poorly metastasizing CC531s cell. The m2-cell line was relatively resistant to killing by NK cells. Both CD95L and TRAIL mediated apoptosis was decreased in the m2-cell line. Now, by flow cytometrical analysis of intra- and extra-cellular expressed receptors, we show that the localization of the receptors for CD95L and TRAIL was not altered in the CC531s-m2 cells as compared to the parental cell line. Subsequently caspase-activation and mitochondrial function were studied by enzymatic cleavage of fluorescent caspase-substrates and retention of the mitochondrial dye rhodamine-123, respectively. The activation of caspases as well as the loss of the mitochondrial membrane potential (MMP) was less in the CC531s-m2 cell line upon CD95L- and TRAIL-signalling. Furthermore, the sensitivity of the CC531-m2 towards cisplatin-induced apoptosis was strongly decreased. This was consistent with less mitochondrial damage, delayed caspase cleavage and decreased caspase activity. Altogether, we conclude that an Natural Killer-cell insensitive cell is less sensitive to CD95L- and TRAIL-induced apoptosis as well as anti-cancer drug induced apoptosis by prevention of mitochondrial damage and activation of caspases.