Hak-Bong Kim

TRAIL sensitize MDR cells to MDR-related drugs by down-regulation of P-glycoprotein through inhibition of DNA-PKcs/Akt/GSK-3β pathway and activation of caspases

BackgroundThe development of new modulator possessing high efficacy, low toxicity and high selectivity is a pivotal approach to overcome P-glycoprotein (P-gp) mediated multidrug resistance (MDR) in cancer treatment. In this study, we suggest a new molecular mechanism that TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) down-regulates P-glycoprotein (P-gp) through inhibition of DNA-PKcs/Akt/GSK-3β pathway and activation of caspases and thereby sensitize MDR cells to MDR-related drugs.ResultsMDR variants, CEM/VLB10-2, CEM/VLB55-8 and CEM/VLB100 cells, with gradually increased levels of P-gp derived from human lymphoblastic leukemia CEM cells, were gradually more susceptible to TRAIL-induced apoptosis and cytotoxicity than parental CEM cells. The P-gp level of MDR variants was positively correlated with the levels of DNA-PKcs, pAkt, pGSK-3β and c-Myc as well as DR5 and negatively correlated with the level of c-FLIPs. Hypersensitivity of CEM/VLB100 cells to TRAIL was accompanied by the activation of mitochondrial apoptotic pathway as well as the activation of initiator caspases. In addition, TRAIL-induced down-regulation of DNA-PKcs/Akt/GSK-3β pathway and c-FLIP and up-regulation of cell surface expression of death receptors were associated with the increased susceptibility to TRAIL of MDR cells. Moreover, TRAIL inhibited P-gp efflux function via caspase-3-dependent degradation of P-gp as well as DNA-PKcs and subsequently sensitized MDR cells to MDR-related drugs such as vinblastine and doxorubicin. We also found that suppression of DNA-PKcs by siRNA enhanced the susceptibility of MDR cells to vincristine as well as TRAIL via down-regulation of c-FLIP and P-gp expression and up-regulation of DR5.ConclusionThis study showed for the first time that the MDR variant of CEM cells was hypersensitive to TRAIL due to up-regulation of DR5 and concomitant down-regulation of c-FLIP, and degradation of P-gp and DNA-PKcs by activation of caspase-3 might be important determinants of TRAIL-induced sensitization of MDR cells to MDR-related drugs. Therefore, combination of TRAIL and chemotherapeutic drugs may be a good strategy for treatment of cancer with multidrug resistance.

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.

Cotreatment with apicidin overcomes TRAIL resistance via inhibition of Bcr-Abl signaling pathway in K562 leukemia cells.

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal cells. Although many cancer cells are sensitive to TRAIL-induced apoptosis, chronic myeloid leukemia (CML) develops resistance to TRAIL. In this study, we investigated whether apicidin, a novel histone deacetylase inhibitor, could overcome the TRAIL resistance in CML-derived K562 cells. Compared to treatment with apicidin or TRAIL alone, cotreatment with apicidin and TRAIL-induced apoptosis synergistically in K562 cells. This combination led to activation of caspase-8 and Bcl-2 interacting domain (Bid), resulting in the cytosolic accumulation of cytochrome c from mitochondria as well as an activation of caspase-3. Treatment with apicidin resulted in down-regulation of Bcr-Abl and inhibition of its downstream target, PI3K/AKT-NF-kappaB pathway. In addition, apicidin decreased the level of NF-kappaB-dependent Bcl-x(L), leading to caspase activation and Bid cleavage. These results suggest that apicidin may sensitize K562 cells to TRAIL-induced apoptosis through caspase-dependent mitochondrial pathway by regulating expression of Bcr-Abl and its related anti-apoptotic proteins. Therefore, the present study suggests that combination of apicidin and TRAIL may be an effective strategy for treating TRAIL-resistant Bcr-Abl expressing CML cells.

Amurensin G, a novel SIRT1 inhibitor, sensitizes TRAIL-resistant human leukemic K562 cells to TRAIL-induced apoptosis

Many types of cancer cells remain resistant towards TRAIL-induced cytotoxicity by the blockade of apoptotic signaling cascades. Thus, sensitizers are needed to enhance the effect of TRAIL-based cancer therapies. Although synergistic tumor cell death has been reported when various HDAC inhibitors were administered with TRAIL in a variety of human cancers, the effect of inhibitors of Class III HDAC such as SIRT1 have not been reported. We reported here for the first time that inhibition of SIRT1 augmented the cytotoxic and apoptotic effects of TRAIL on human leukemic K562 cells. Knockdown of SIRT1 or treatment with amurensin G, a potent new SIRT1 inhibitor, up-regulated the levels of DR5 and c-Myc and down-regulated the level of c-FLIP(L/S). Furthermore, knockdown of SIRT1 or treatment with amurensin G augmented the molecular responses to TRAIL, including activation of caspase-8, -9 and -3, PARP cleavage, up-regulation of Bax, and down-regulation of Bcl-2. Amurensin G-enhanced TRAIL-induced apoptosis was abrogated by caspase inhibitor Z-VAD-FMK. These findings suggest that the suppression of SIRT1 with siRNA or amurensin G sensitize the TRAIL-resistant K562 cell to TRAIL-induced apoptosis, possibly by the up-regulation of c-Myc and DR5 surface expression and the down-regulations of c-FLIP and Mcl-1. In addition, amurensin G, a potent new SIRT1 inhibitor, would be used as a sensitizer of TRAIL in TRAIL-resistant leukemic cells.

Suppression of multidrug resistance by treatment with TRAIL in human ovarian and breast cancer cells with high level of c-Myc

In this study, we investigated the role of c-Myc in overcoming multidrug resistance (MDR) in human ovarian and breast cancer cells by TRAIL. We showed that P-gp expressing MDR variants (Hey A8-MDR and MCF7-MDR cells) with high level of c-Myc were highly susceptible to TRAIL treatment when compared to their drug-sensitive parental human ovarian cancer Hey A8 and breast MCF-7 cells, respectively. Up-regulation of DR5 TRAIL receptor and down-regulation of c-FLIP and the promotion of caspase-dependent cell death, which contribute to TRAIL sensitization of MDR cells, were regulated by the over-expressed c-Myc in the MDR cells. After targeted inhibition of c-Myc with specific siRNA, these responses to TRAIL disappeared and TRAIL-induced apoptosis was also suppressed in MCF7-MDR cells. Treatment with TRAIL significantly reduced P-glycoprotein (P-gp)-mediated efflux of rhodamine123 in both Hey A8-MDR and MCF7-MDR cells. Furthermore, TRAIL significantly potentiated the cytotoxicity of vinblastine, vincristine, doxorubicin and VP-16 that are P-gp substrate anticancer drugs in both MDR cells, which resulted in the reversal effect of TRAIL on the MDR phenotype. The present study shows for the first time that elevated c-Myc expression in the MDR cells plays a critical role in overcoming MDR by TRAIL that can act as a specific sensitizer for P-gp substrate anticancer drug.

Sensitization of human K562 leukemic cells to TRAIL-induced apoptosis by inhibiting the DNA-PKcs/Akt-mediated cell survival pathway

Despite the fact that many cancer cells are sensitive to TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, human K562 leukemic cells showed resistance to TRAIL-induced apoptosis. Interestingly, K562/R3 cells, a stable TRAIL-sensitive variant isolated from K562 cells, showed down-regulation of DNA-PK/Akt pathway and a high responsiveness to TRAIL-mediated growth inhibition and apoptosis. We revealed that siRNA-mediated suppression of DNA-PKcs led to decreased phosphorylation of Akt and Bad, a target molecule of Akt, and increased expression of DR4/DR5. Also, we found that suppression of DNA-PKcs using siRNA down-regulated c-FLIP and sensitized K562 cells to TRAIL-induced apoptosis through activation of caspase-8, -9 and -3. In addition, we revealed that treatment with DMNB, a specific inhibitor of DNA-PK, resulted in an increase of DR4/DR5 mRNA levels and their surface expression and a decrease of c-FLIP mRNA levels in K562 cells. DMNB potentiated TRAIL-induced cytotoxicity and apoptosis through inhibition of DNA-PK/Akt pathway and activation of caspase-8, -9 and -3 in K562 cells. This study is the first to show that a protective role of DNA-PK/Akt pathway against TRAIL-induced apoptosis and thus TRAIL in combination with agents that inhibit DNA-PK/Akt pathway would have clinical applicability in treating TRAIL-insensitive human leukemic cells. This model may provide a novel framework for overcoming TRAIL resistance of other cancer cells with agents that inhibit DNA-PK/Akt pathway.

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and β-catenin and hypoxic preconditioning effect of polyphenols

SIRT1 has been found to function as a Class III deacetylase that affects the acetylation status of histones and other important cellular nonhistone proteins involved in various cellular pathways including stress responses and apoptosis. In this study, we investigated the role of SIRT1 signaling in the hypoxic down-regulations of c-Myc and β-catenin and hypoxic preconditioning effect of the red wine polyphenols such as piceatannol, myricetin, quercetin and resveratrol. We found that the expression of SIRT1 was significantly increased in hypoxia-exposed or hypoxic preconditioned HepG2 cells, which was closely associated with the up-regulation of HIF-1α and down-regulation of c-Myc and β-catenin expression via deacetylation of these proteins. In addition, blockade of SIRT1 activation using siRNA or amurensin G, a new potent SIRT1 inhibitor, abolished hypoxia-induced HIF-1α expression but increased c-Myc and β-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, β-catenin and PHD2 under hypoxia. We also found that myricetin, quercetin, piceatannol and resveratrol up-regulated HIF-1α and down-regulated c-Myc, PHD2 and β-catenin expressions via SIRT1 activation, in a manner that mimics hypoxic preconditioning. This study provides new insights of the molecular mechanisms of hypoxic preconditioning and suggests that polyphenolic SIRT1 activators could be used to mimic hypoxic/ischemic preconditioning.

Sensitization of imatinib-resistant CML cells to TRAIL-induced apoptosis is mediated through down-regulation of Bcr-Abl as well as c-FLIP.

Resistance to imatinib is commonly associated with reactivation of Bcr-Abl signalling. However, Bcr-Abl-independent signalling pathways may be activated and contributed to imatinib resistance in some CML (chronic myelogenous leukaemia) patients. We had isolated three imatinib-resistant K562/R1, R2 and R3 variants with gradual loss of Bcr-Abl from K562 cells to develop effective therapeutic strategies for imatinib-resistant CML. Interestingly, we found that these cells became highly sensitive to TRAIL (tumour necrosis factor-related apoptosis-inducing factor) in comparison with K562 cells showing high resistance to TRAIL. Treatment of K562/R3 cells with TRAIL resulted in activation of TRAIL receptor pathway by including caspase 8 activation, Bid cleavage, cytochrome c release and caspase 3 activation. These results were accompanied by down-regulation of c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-inhibitory protein} in imatinib-resistant K562 variants compared with K562 cells. Overexpression of c-FLIP in K562/R3 cells acquired TRAIL resistance and conversely, c-FLIP-silenced K562 cells became sensitive to TRAIL. Moreover, Bcr-Abl-silenced K562 cells showed down-regulation of c-FLIP and the subsequent overcome of TRAIL resistance. Taken together, our results demonstrated for the first time that the loss of Bcr-Abl in imatinib-resistant cells led to the down-regulation of c-FLIP and subsequent increase of TRAIL sensitivity, suggesting that TRAIL could be an effective strategy for the treatment of imatinib-resistant CML with loss of Bcr-Abl.

Sensitization of chemo-resistant human chronic myeloid leukemia stem-like cells to Hsp90 inhibitor by SIRT1 inhibition.

Development of effective therapeutic strategies to eliminate cancer stem-like cells (CSCs), which play a major role in drug resistance and disease recurrence, is critical to improve cancer treatment outcomes. The current investigation was undertaken to examine the effectiveness of the combination treatment of Hsp90 inhibitor and SIRT1 inhibitor in inhibiting the growth of chemo-resistant stem-like cells isolated from human chronic myeloid leukemia K562 cells. Inhibition of SIRT1 by use of SIRT1 siRNA or SIRT1 inhibitors (amurensin G and EX527) effectively potentiated sensitivity of Hsp90 inhibitors (17-AAG and AUY922) in CD44high K562 stem-like cells expressing high levels of CSC-related molecules including Oct4, CD34, β-catenin, c-Myc, mutant p53 (mut p53), BCRP and P-glycoprotein (P-gp) as well as CD44. SIRT1 depletion caused significant down-regulation of heat shock factor 1 (HSF1)/heat shock proteins (Hsps) as well as these CSC-related molecules, which led to the sensitization of CD44high K562 cells to Hsp90 inhibitor by SIRT1 inhibitor. Moreover, 17-AAG-mediated activation of HSF1/Hsps and P-gp-mediated efflux, major causes of Hsp90 inhibitor resistance, was suppressed by SIRT1 inhibitor in K562-CD44high cells. Our data suggest that combined treatment with Hsp90 inhibitor and SIRT1 inhibitor could be an effective therapeutic approach to target CSCs that are resistant to current therapies.

Design of core–shell magnetic mesoporous silica hybrids for pH and UV light stimuli-responsive cargo release

This paper reports a controlled drug release strategy through guest diffusion pathways with a “three-in-one” system by combining three advantages together in a single entity. The designed system is composed of superparamagnetic Fe3O4 nanoparticles as the core, a mesoporous silica hybrid as the shell with specific functional moieties, and external trigger (UV-light) responsive functional derivatives as the nanoregulators. The magnetic mesoporous silica hybrid nanospheres (MSH@Azo-CA) were quite responsive to external stimuli such as (i) UV-light and (ii) pH-triggered drug release. The core–shell mesoporous silica hybrid nanospheres were used as a drug carrier for the loading and controlled release of model cargo (e.g. doxorubicin hydrochloride (DOX)/Rh123), because of its combined external (UV-light) and internal (cellular pH) stimuli-responsive behavior and biocompatibility. The experimental study showed that the drug release behavior depends mainly on the UV-light (365 nm)-actuated ‘trans’ conformation and ‘cis’ conformation of the nanoregulators (chrysoidine derivatives) and the intracellular pH of the release medium. The presence of external and internal triggers can result in the good controlled release of loaded cargoes to the target sites. The cytotoxicity of the synthesized core–shell hybrid mesoporous silica nanospheres was examined using MCF-7 cells. The intracellular uptake and release process was observed by confocal laser scanning microscopy (CLSM). In addition, the presence of a magnetic core means that these silica hybrid nanospheres have potential use in the efficient targeted delivery of anticancer agents that can be directed by an external magnetic field for the delivery with a dose predetermined by the ‘ON’ and ‘OFF’ command driven by the external UV-light trigger.