Soo-Jung Park

Down-regulation of FoxO-dependent c-FLIP expression mediates TRAIL-induced apoptosis in activated hepatic stellate cells.

Activated hepatic stellate cells which contribute to liver fibrosis have represented an important target for antifibrotic therapy. In this study, we found that TRAIL inhibited PI3K/Akt-dependent FoxO phosphorylation and relocated FoxO proteins into the nucleus from the cytosol in activated human hepatic stellate LX-2 cells. The accumulated FoxO proteins in the nucleus led to down-regulation of c-FLIP(L/S) expression, resulting in the activation of apoptosis-related signaling molecules including the activation of caspase-8, -3, and Bid, as well as mitochondrial cytochrome c release. These results were supported by showing that siRNA-mediated knockdown of FoxO led to restoration of c-FLIP(L/S) expression and resistance to TRAIL-induced apoptosis after treatment of LX-2 cells with TRAIL. Furthermore, c-FLIP(L/S)-transfected LX-2 cells showed the decreased sensitivity to TRAIL-induced apoptosis. Collectively, our data suggest that sequential activation of FoxO proteins under conditions of suppressed PI3K/Akt signaling by TRAIL can down-regulate c-FLIP(L/S), consequently promoting TRAIL-induced apoptosis in LX-2 cells. Therefore, the present study suggests TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.

Quercetin enhances susceptibility to NK cell-mediated lysis of tumor cells through induction of NKG2D ligands and suppression of HSP70.

It is known that treatments with heat shock, some anticancer drugs, and ionizing radiation increase the expression of heat-shock proteins (HSPs) and natural killer group 2D (NKG2D) ligands in tumor cells. The increased HSPs may make the tumor cells resistant to apoptosis and reduction of HSPs may make the tumor cells more susceptible to natural killer (NK)-cell mediated lysis of tumor cells. In this study, we investigated whether quercetin which has inhibitory activities against heat-shock factor, protein kinase C, nuclear factor-κB, and phosphatidyl inositol 3-kinase, can modulate the expression of NKG2D ligands and suppress the HSPs in tumor cells. The results of this study showed that quercetin significantly induced the expression of several NKG2D ligands including major histocompatibility complex class I-related chain B, UL16-binding protein 1, and UL16-binding protein 2 in K562, SNU1, and SNU-C4 cells. The quercetin-treated K562, SNU1, and SNU-C4 cells showed an enhanced susceptibility to NK-92 cells through induction of NKG2D ligands. This increased expression of NKG2D ligands seemed to be due to the inhibition of the nuclear factor-κB and phosphatidyl inositol 3-kinase pathways. The findings of this study suggest that the induced NKG2D ligands with the decrease of HSP70 protein by quercetin may provide an attractive strategy to improve the effectiveness of NK cell-based cancer immunotherapy.

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.

EGFR Inhibitors Enhanced the Susceptibility to NK Cell-mediated Lysis of Lung Cancer Cells

As quercetin, which can inhibit phosphatidylinositol 3-kinase, nuclear factor-kappa B, and protein kinase C (PKC) pathways, induced expression of natural killer group 2, member D (NKG2D) ligands on cancer cells and made the cells sensitive to NK –cell-mediated killing; inhibition of epidermal growth factor receptor (EGFR) pathway might lead to induction of NKG2D ligands. In this study, it was investigated whether EGFR inhibitors, including erlotinib or gefitinib, could regulate expression of NKG2D ligands in various lung cancer cells including A549, NCI-H23, and SW-900. The EGFR inhibitors predominantly increased transcription and surface expression of ULBP1, and subsequently increased susceptibility of the cancer cells to NK-92 cells. When the selective inhibitors of nuclear factor-kappa B, phosphatidylinositol 3-kinase, mitogen-activated protein kinases, and PKC were treated to discriminate downstream signaling of EGFR pathway, expression of ULBP1 in the cancer cells was induced by inhibition of PKC. Treatment with phorbol 12-myristate 13-acetate restored the EGFR inhibitor-induced ULBP1 transcription. Binding activity to ULBP1 promoter region of AP-2&agr;, which suggested as suppressor of expression of ULBP1, was decreased by treatment with EGFR inhibitors, and restored by pretreatment with phorbol 12-myristate 13-acetate in A549 and SW-900. Rottlerin, a PKC&dgr; inhibitor, also decreased the binding activity of AP-2&agr; in dose-dependent manner. This study suggests that EGFR inhibitors enhanced the susceptibility to NK cell-mediated lysis of lung cancer cells by induction of ULBP1 by inhibition of PKC pathway and therapeutic efficacy of EGFR inhibitors in lung cancer may be mediated in part by increased susceptibility to NK cell-mediated cytotoxicity.

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.

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.

TRAIL regulates collagen production through HSF1-dependent Hsp47 expression in activated hepatic stellate cells

Hsp47 is a collagen-specific molecular chaperone, whose activity has been implicated in liver fibrosis. In this study, we showed that TRAIL treatment inhibited Hsp47 expression in dose- and time-dependent manners, subsequently leading to the decrease of collagen production in activated human hepatic stellate LX-2 cells. Overexpression of Hsp47 in LX-2 cells acquired resistance for TRAIL-induced collagen reduction and conversely, siRNA suppression of Hsp47 enhanced the decrease of collagen production due to TRAIL treatment. Moreover, we found that Hsp47 expression was under the transcriptional control of heat shock factor (HSF) 1 which is highly located on nucleus in activated human hepatic stellate LX-2 cells. Treatment of LX-2 cells with TRAIL decreased the active trimer formation of HSF1, increased the dephosphorylation of HSF1 (Ser(230)), and enhanced the translocation of HSF1 into cytosol. The accumulated HSF1 in cytosol led to downregulation of Hsp47 expression, resulting in the reduction of collagen production. Consistently, HSF1 silencing by siRNA prevented Hsp47 induction and subsequent collagen production, whereas overexpression of HSF1 restored the expression level of Hsp47 as well as collagen production in response to TRAIL treatment in LX-2 cells. Taken together, our data suggested that TRAIL induced HSF1 inactivation, consequently leading to the suppression of Hsp47-dependent collagen production in activated human hepatic stellate cells. Therefore, this study suggests that TRAIL may be an effective strategy for antifibrotic therapy in liver fibrosis.

Direct fabrication of thin film gold resistance temperature detection sensors on a curved surface using a flexible dry film photoresist and their calibration up to 450 °C

High efficiency heat exchangers, such as intercoolers and recuperators, are composed of complex and compact structures to enhance heat transfer. This limits the installation of conventional temperature sensors to measure the temperature inside the heat exchanger without flow disturbance. To overcome this limitation, we have developed a direct patterning method in which metal is sputtered onto a curved surface using film photoresist and the fabrication of thin film Au resistance temperature detection (RTD) temperature sensors. A photosensitive film resist has been used to overcome the difficulty of 3-dimensional photolithography on a curved surface. The film resist after 2-dimensional photolithography is laminated over an alumina rod which is deposited with Au as an RTD sensing material. The Au metal is etched chemically, and the film resist is removed to form the thin film Au-RTD temperature sensors. They are calibrated by measuring the resistance change against temperature in a thermally controlled furnace. The second order polynomial fit shows good agreement with the measured temperatures with a standard deviation of 0.02 for the temperature range of 20–450 °C. Finally, the performance of the Au-RTD temperature sensors was evaluated.

Quercetin Potentiates TRAIL-induced Apoptosis in Human Colon KM12 Cells

Many cancer cells are sensitive to the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, some cancer cells show either partial or complete resistance to TRAIL. Human colon carcinoma KM12 cells have been shown to be insensitive to TRAIL-induced apoptosis. To overcome TRAIL resistance in KM12 cells, we targeted key anti-apoptotic molecules involved in the modulation of TRAIL resistance in the cells, and evaluated the effects of quercetin as a TRAIL sensitizer in the cells. We found that quercetin acted in synergy with TRAIL to enhance TRAIL-induced apoptosis in KM12 cells by the down-regulation of c-FLIP and DNA-PKcs/Akt and up-regulation of death receptors (DR4/DR5), which led to the enhancement of TRAIL-mediated activation of caspases and subsequent cleavage of PARP, as well as up-regulation of Bax. These findings suggest that the DNA-PKcs/Akt signaling pathway, as well as c-FLIP, play essential roles in regulating cells in the escape from TRAIL-induced apoptosis. Based on these results, this study provides a potential application of quercetin in combination with TRAIL in the treatment of human colon cancer.