Downregulation of NKG2DLs by TGF-ß in human lung cancer cells.

BACKGROUND: Transforming growth factor beta (TGF-ß) is a typical immuno-inhibitory cytokine and highly secreted by lung cancer cells. It was supposed that its immunosuppressive effects to NK cell might be related with the altered expression of activating and inhibitory molecules in lung cancer cells. In this study, we examined the expression of NKG2DLs, PD-L1 and PD-L2 in lung cancer cells after treatment of TGF-ß and a TGF-ß inhibitor, Galunisertib (LY2157299). RESULTS: TGF-ß reduced the level of surface proteins of five NKG2DLs without altered transcription levels in lung cancer cells. Galunisertib reversed the effect of TGF-ß on the expression of NKG2DLs. Since MMP inhibitors, MMPi III and MMP2 inhibitor I, restored the reduced expression of NKG2DLs after treatment of TGF-ß, it was thought that TGF-ß induced the expression of MMP2 which facilitated the shedding of the NKG2DLs in cancer cells. However, the expression of PD-L1, L2 were not changed by treatment with TGF-ß or Galunisertib. CONCLUSIONS: Therefore, inhibition of TGF-ß might reverse the immunosuppressive status on immune cells and restore NK cell mediated anticancer immune responses by upregulation of NKG2DLs in cancer cells.

Differential Effects of Histone Deacetylases on the Expression of NKG2D Ligands and NK Cell-Mediated Anticancer Immunity in Lung Cancer Cells.

Histone acetylation is an epigenetic mechanism that regulates the expression of various genes, such as natural killer group 2, member D (NKG2D) ligands. These NKG2D ligands are the key molecules that activate immune cells expressing the NKG2D receptor. It has been observed that cancer cells overexpress histone deacetylases (HDACs) and show reduced acetylation of nuclear histones. Furthermore, HDAC inhibitors are known to upregulate the expression of NKG2D ligands. Humans have 18 known HDAC enzymes that are divided into four classes. At present, it is not clear which types of HDAC are involved in the expression of NKG2D ligands. We hypothesized that specific types of HDAC genes might be responsible for altering the expression of NKG2D ligands. In this study, we monitored the expression of NKG2D ligands and major histocompatibility complex (MHC) class I molecules in lung cancer cells which were treated with six selective HDAC inhibitors and specific small interfering RNAs (siRNAs). We observed that treatment with FK228, which is a selective HDAC1/2 inhibitor, also known as Romidepsin, induced NKG2D ligand expression at the transcriptional and proteomic levels in two different lung cancer cell lines. It also caused an increase in the susceptibility of NCI-H23 cells to NK cells. Silencing HDAC1 or HDAC2 using specific siRNAs increased NKG2D ligand expression. In conclusion, it appears that HDAC1 and HDAC2 might be the key molecules regulating the expression of NKG2D ligands. These results imply that specifically inhibiting HDAC1 and HDAC2 could induce the expression of NKG2D ligands and improve the NK cell-mediated anti-cancer immunity.

COX-2- and endoplasmic reticulum stress-independent induction of ULBP-1 and enhancement of sensitivity to NK cell-mediated cytotoxicity by celecoxib in colon cancer cells.

In the present study, we investigated whether celecoxib could induce the expression of NKG2D ligands in clonogenic colon cancer cells, and increase their susceptibility to NK cell-mediated cell death. Celecoxib and its non-coxib analog, 2,5-dimethyl celecoxib, induced ULBP-1 and DR5 in both COX-2 negative HCT-15 cells and COX-2 positive HT-29 cells. Celecoxib increased their susceptibility to NK92 cells in both DELFIA assay and soft agar colony forming assay. The inducibility of ULBP-1 and DR5 by celecoxib was not different between CD44- and CD44+ HCT-15 cells, and CD133- and CD133+ HT-29 cells. Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells, at least comparable to less clonogenic CD44- HCT-15 and CD133- HT-29 cells, respectively. In addition, celecoxib induced CHOP, and thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5 but not ULBP1 in HCT-15. Taken together, these findings suggest that celecoxib induces the expression of ULBP-1 as well as DR5 in clonogenic colon cancer cells via COX-2 and ER stress-independent pathways, and increases their susceptibility to NK cells.

Combination of cancer immunotherapy with clinically available drugs that can block immunosuppressive cells.

Although cancer immunotherapy, which is able to target specifically cancer cells without detrimental effects to normal cell functions, would serve as an ideal therapeutic modality, most of the randomized clinical trials of cancer immunotherapy have not demonstrated a meaningful survival benefit to cancer patients over preexisting therapeutic modalities. Due to the discrepancy between the impressive preclinical results and the limited clinical results, the cancer immunotherapy is not accepted generally as a standard therapy for cancers. A variety of immune escape mechanisms are thought to be involved in this ineffectiveness of cancer immunotherapy. Therefore, elimination of immunosuppressive activities in tumor microenvironment will enhance the effectiveness of cancer immunotherapy, which is currently focused on activation of tumor-specific immune responses. Since there are now increasing evidences showing that many cytotoxic anticancer drugs including targeted agents given in lower-than-therapeutic doses have not only the ability to eliminate tumor cells but can also block the immunosuppressive activities in tumor microenvironments and consequently favor the development of anticancer immune responses, clinically available drugs can be considered for their rapid application to cancer immunotherapies to enhance the efficacy of cancer immunotherapies with marginal effects on cancer treatment.

Amurensin G enhances the susceptibility to tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity of cancer stem-like cells of HCT-15 cells.

Cancer stem cells (CSCs) are resistant to radiotherapy and chemotherapy and play a significant role in cancer recurrence. Design of better treatment strategies that can eliminate or otherwise control CSC populations in tumors is necessary. In this study, the sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity and the effect of amurensin G, a novel sirtuin 1 (SIRT1) inhibitor, were examined using the CSC-enriched fraction of HCT-15 human colon cancer cells. Cancer stem cell-enriched HCT-15 colony cells were paradoxically less sensitive to doxorubicin, and more sensitive to TRAIL-induced cytotoxicity, than their parental cells. Also, CD44(+) HCT-15 cells were more susceptible to TRAIL-mediated cytotoxicity than CD44(-) HCT-15 cells, possibly due to increased levels of death receptors DR4 and DR5 as well as c-Myc, and decreased levels of c-FLIPL /S in CD44(+) cells compared with CD44(-) HCT-15 cells. The combination effect of amurensin G on TRAIL-mediated cytotoxicity was much more apparent in CD44(+) cells than in CD44(-) HCT-15 cells, and this was associated with more prominent downregulation of c-FLIP(L/S) in CD44(+) cells than in CD44(-) HCT-15 cells. These results indicate that HCT-15 colony or CD44(+) cells, which may have CSC properties, are more sensitive to TRAIL than parental or CD44(-) HCT-15 cells. Amurensin G may be effective in eliminating colon CSCs and be applicable to potentiate the sensitivity of colon CSCs to TRAIL.

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.

Susceptibility to natural killer cell-mediated lysis of colon cancer cells is enhanced by treatment with epidermal growth factor receptor inhibitors through UL16-binding protein-1 induction.

We have previously shown that inhibition of intracellular signaling pathways by treatment with quercetin induced the expression of natural killer cell group 2D (NKG2D) ligands on cancer cells and made the cells sensitive to natural killer (NK)-cell mediated cytotoxicity. In the present study, we investigated whether epidermal growth factor receptor (EGFR) inhibitors could induce the expression of NKG2D ligands in colon cancer cells. Treatment with EGFR inhibitors predominantly increased the levels of mRNA transcripts and surface protein of UL16-binding protein-1 (ULBP1) in various colon cancer cells, including KM12, Caco-2, HCT-15, and HT-29, which express EGFR, and increased susceptibility of these colon cancer cells to NK-92 cells. The expression of ULBP1 was not induced by inhibitors of nuclear factor-κB, phosphatidylinositol 3 kinase, and MAPK, but was induced by inhibitors of PKC, and the induction of ULBP1 expression with EGFR inhibitors was prevented by treatment with PMA in colon cancer cells. A transcription factor, activator protein-2 alpha (AP-2α), which has a suppressive effect on ULBP1 transcription, was prevented from binding to the ULBP1 promoter by treatment with EGFR inhibitors. The present study suggests that EGFR inhibitors can enhance the susceptibility to NK cell-mediated lysis of colon cancer cells by induction of ULBP1 via inhibition of the PKC pathway.

Involvement of SIRT1 in hypoxic down-regulation of c-Myc and ß-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 ß-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 ß-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 ß-catenin expression. SIRT1 was also found to stabilize HIF-1α protein and destabilize c-Myc, ß-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 ß-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.

Cancer spheres from gastric cancer patients provide an ideal model system for cancer stem cell research.

Cancer stem cells have been hypothesized to drive the growth and metastasis of tumors. Because they need to be targeted for cancer treatment, they have been isolated from many solid cancers. However, cancer stem cells from primary human gastric cancer tissues have not been isolated as yet. For the isolation, we used two cell surface markers: the epithelial cell adhesion molecule (EpCAM) and CD44. When analyzed by flow cytometry, the EpCAM(+)/CD44(+) population accounts for 4.5% of tumor cells. EpCAM(+)/CD44(+) gastric cancer cells formed tumors in immunocompromised mice; however, EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) and EpCAM(-)/CD44(+) cells failed to do so. Xenografts of EpCAM(+)/CD44(+) gastric cancer cells maintained a differentiated phenotype and reproduced the morphological and phenotypical heterogeneity of the original gastric tumor tissues. The tumorigenic subpopulation was serially passaged for several generations without significant phenotypic alterations. Moreover, EpCAM(+)/CD44(+), but not EpCAM(-)/CD44(-), EpCAM(+)/CD44(-) or EpCAM(-)/CD44(+) cells grew exponentially in vitro as cancer spheres in serum-free medium, maintaining the tumorigenicity. Interestingly, a single cancer stem cell generated a cancer sphere that contained various differentiated cells, supporting multi-potency and self-renewal of a cancer stem cell. EpCAM(+)/CD44(+) cells had greater resistance to anti-cancer drugs than other subpopulation cells. The above in vivo and in vitro results suggest that cancer stem cells, which are enriched in the EpCAM(+)/CD44(+) subpopulation of gastric cancer cells, provide an ideal model system for cancer stem cell research.

High susceptibility of metastatic cells derived from human prostate and colon cancer cells to TRAIL and sensitization of TRAIL-insensitive primary cells to TRAIL by 4,5-dimethoxy-2-nitrobenzaldehyde.

BACKGROUND: Tumor recurrence and metastasis develop as a result of tumors' acquisition of anti-apoptotic mechanisms and therefore, it is necessary to develop novel effective therapeutics against metastatic cancers. In this study, we showed the differential TRAIL responsiveness of human prostate adenocarcinoma PC3 and human colon carcinoma KM12 cells and their respective highly metastatic PC3-MM2 and KM12L4A sublines and investigated the mechanism underlying high susceptibility of human metastatic cancer cells to TRAIL. RESULTS: PC3-MM2 and KM12L4A cells with high level of c-Myc and DNA-PKcs were more susceptible to TRAIL than their poorly metastatic primary PC3 and KM12 cells, which was associated with down-regulation of c-FLIPL/S and Mcl-1 and up-regulation of the TRAIL receptor DR5 but not DR4 in both metastatic cells. Moreover, high susceptibility of these metastatic cells to TRAIL was resulted from TRAIL-induced potent activation of caspase-8, -9, and -3 in comparison with their primary cells, which led to cleavage and down-regulation of DNA-PKcs. Knockdown of c-Myc gene in TRAIL-treated PC3-MM2 cells prevented the increase of DR5 cell surface expression, caspase activation and DNA-PKcs cleavage and attenuated the apoptotic effects of TRAIL. Moreover, the suppression of DNA-PKcs level with siRNA in the cells induced the up-regulation of DR5 and active caspase-8, -9, and -3. We also found that 4,5-dimethoxy-2-nitrobenzaldehyde (DMNB), a specific inhibitor of DNA-PK, potentiated TRAIL-induced cytotoxicity and apoptosis in relatively TRAIL-insensitive PC3 and KM12 cells and therefore functioned as a TRAIL sensitizer. CONCLUSION: This study showed the positive relationship between c-Myc expression in highly metastatic human prostate and colon cancer cells and susceptibility to TRAIL-induced apoptosis and therefore indicated that TRAIL might be used as an effective therapeutic modality for advanced metastatic cancers overexpressing c-Myc and combination of TRAIL therapy with agent that inhibits the DNA-PKcs/Akt signaling pathway might be clinically useful for the treatment of relatively TRAIL-insensitive human cancers.

Induction of NKG2D ligands and increased sensitivity of tumor cells to NK cell-mediated cytotoxicity by hematoporphyrin-based photodynamic therapy.

Natural killer (NK) cells are important innate effector cells which can irradicate tumor cells through specific interactions between activating receptors on NK cells and their cognate ligands on cancer cells. Recently, it has been known that induction of activating NKG2D ligands including MHC class I chain-related (MIC) and UL16-binding protein (ULBP) families on tumor cells by various stresses makes them more susceptible to NK cell-mediated cytotoxicity. Therefore, it was investigated whether sublethal dose of hematoporphyrin-based photodynamic therapy (PDT) could up-regulate NKG2D ligands on tumor cells and increase the susceptibility of cancer cells against NK cells. Treatment with sublethal dose of hematoporphyrin-based PDT increased mRNA transcription and surface expression of ULBP1 and ULBP2 genes in SNU-1 human gastric tumor cell line and MICA/B, ULBP1, ULBP2 and ULBP3 genes in SW-900 human lung cancer cell line. These results were followed by increased susceptibility of cancer cells to NK cell-mediated cytotoxicity after sublethal PDT, which was abolished by addition of a blocking NKG2D mAb. Therefore, it could be suggested that the effect of hematoporphyrin-based PDT might be mediated in part by the increased susceptibility to NK cells via induction of NKG2D ligands on tumor cells, which survived after treatment with PDT.

Cyclophosphamide potentiates the antitumor effect of immunization with injection of immature dendritic cells into irradiated tumor.

Growth of a tumor on the left flank was suppressed by direct injection of immature DCs (iDCs) into the irradiated tumor on the right thigh (IR/DC). This antitumor immune effect of IR/DC was enhanced by pretreatment with CTX (CTX+IR/DC) and this effect was related with increased number of tumor-specific IFN-γ secreting T cells and decreased ratio of CD4(+)CD25(+)/CD4(+) T cells. The treatment with CTX+IR/DC increased or decreased the levels of IL-2 or IL-10, respectively. These results demonstrated that antitumor effect of IR/DC could be augmented by pretreatment with low-dose CTX, suggesting a new antitumor therapeutic modality of chemoradioimmunotherapy.

An inhibitor of programmed death ligand 1 enhances natural killer cell-mediated immunity against malignant melanoma cells.

Since ionizing radiation has showed the dramatic effect to kill the cancer cells through direct DNA damage as well as triggering anti-cancer immune responses including induction of NKG2D ligands, it has used for long time to treat many cancer patients. However, it has been known that radiotherapy might promote the remnant cancer cells to escape immune system and metastasis. One of the suggested ways of immune evasion is induction of a ligand for programmed death-1 (PD-L1) in head and neck cancer, bladder cancer and lung cancer cells which engages the receptor, programmed death-1 (PD-1) in immune cells. PD-1/PD-L1 axis transduces the inhibitory signal and suppresses the adaptive immunity. However, their role in innate immunity remains poorly understood. Therefore, we investigated whether ionizing radiation could change the expression of PD-L1 in malignant melanoma cells and the receptor, programmed death-1 (PD-1), in NK-92 cells. Surface PD-L1 levels on melanoma cells were increased by ionizing radiation in a dose-independent manner but the level of PD-L1 was not changed significantly in NK-92 cells. Radiation-induced PD-L1 suppressed the activity of the NK-92 cells against melanoma cells despite of upregulation of NKG2D ligands. Furthermore, activated NK cells had high level of PD-1 and could not kill PD-L1+ melanoma cells effectively. When we used PD-L1 inhibitor or silenced PD-L1 gene, inhibited PD-1/PD-L1 axis reversed the activity of the suppressed NK cells. Through these results, we supposed that PD-1/PD-L1 blockade could enhance the immune responses of NK cells against melanoma cells after radiotherapy and might overcome the PD-L1 mediated radioresistance of cancer cells.

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

BACKGROUND: The 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-3beta pathway and activation of caspases and thereby sensitize MDR cells to MDR-related drugs. RESULTS: MDR 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-3beta 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-3beta 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. CONCLUSION: This 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.

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.

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.

Potentiation of TRAIL­induced cell death by nonsteroidal anti­inflammatory drug in human hepatocellular carcinoma cells through the ER stress­dependent autophagy pathway.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed primary liver malignancy. The limited success with relapse of the disease in HCC therapy is frequently associated with the acquired resistance to anticancer drugs. To develop a strategy and design for overcoming the resistance of HCC cells to TNF­related apoptosis inducing ligand (TRAIL)­induced cell death, we evaluated the efficacy of a non­steroidal anti­inflammatory drug (NSAID) in combination with TRAIL against TRAIL­resistant HCC cells expressing a high level of CD44. We revealed by MTT and western blotting, respectively, that celecoxib (CCB), an NSAID, and 2,5­dimethyl celecoxib (DMC), a non­cyclooxygenase (COX)­2 inhibitor analog of CCB, were able to sensitize TRAIL­resistant HCC cells to TRAIL, implicating a COX­independent mechanism. CCB dose­dependently enhanced LC3­II and reduced p62 levels through AMPK activation and inhibition of the Akt/mTOR pathway and upregulated expression of ATF4/CHOP, leading to activation of endoplasmic reticulum (ER) stress­dependent autophagy. The TRAIL sensitization capacity of CCB in TRAIL­resistant HCC cells was abrogated by an ER stress inhibitor. In addition, we also revealed by flow cytometry and western blotting, respectively, that accelerated downregulation of TRAIL­mediated c­FLIP expression, DR5 activation and CD44 degradation/downregulation by NSAID resulted in activation of caspases and poly(ADP­ribose) polymerase (PARP), leading to the sensitization of TRAIL­resistant HCC cells to TRAIL and thereby reversal of TRAIL resistance. From these results, we propose that NSAID in combination with TRAIL may improve the antitumor activity of TRAIL in TRAIL­resistant HCC, and this approach may serve as a novel strategy that maximizes the therapeutic efficacy of TRAIL for clinical application.

17ß-Estradiol Inhibits Lysophosphatidylcholine-Induced Apoptosis in Cultured Vascular Smooth Muscle Cells.

OBJECTIVES: Coronary heart disease (CHD) risk increases in women after menopause, but menopausal hormone therapy (MHT) helps prevent CHD if started early after menopause. To explore the mechanism underlying the direct vascular actions of estrogen, the effects of 17ß-estradiol (E2) on apoptosis of vascular smooth muscle cells (VSMCs) induced with lysophosphatidylcholine (lysoPC), an active component of oxidized low-density lipoprotein, were investigated in the present study. METHODS: VSMCs were isolated from rat aortas. Apoptosis and protein expression of caspases were assessed using propidium iodide staining and Western blot analysis, respectively. Intracellular formation of reactive oxygen species (ROS) was examined using dichlorofluorescein diacetate, a cell-permeable oxidation-sensitive probe, and quantitated with flow cytometry. Nuclear factor-κB (NF-κB) activation was determined after transfection with a reporter plasmid containing the luciferase reporter gene. RESULTS: After pre-treatment for 24 hours, 17ß-E2 suppressed lysoPC-induced (15 µM) apoptotic cell death in a dose-dependent manner with statistical significance at near physiological concentration. 17ß-E2 (10⁻6 M) also increased protein levels of caspase-9 and -8 precursors and decreased the active form of caspase-3. Western blot analysis using subcellular fractions showed that 17ß-E2 decreased mitochondrial Bax levels and concomitantly increased cytosolic Bax expression. Furthermore, intracellular production of ROS and NF-κB-mediated transcriptional activity were reduced with 17ß-E2. In addition, estrogen effects on apoptosis were partially blocked by ICI 182,780, a specific estrogen receptor antagonist. CONCLUSIONS: In cultured VSMCs treated with lysoPC, 17ß-E2 reduced apoptotic cell death by down-regulating both extrinsic and intrinsic apoptosis pathways, contributing to the preventive action of MHT against CHD.

Effects of 17ß-Estradiol on the Plasminogen Activator System in Vascular Smooth Muscle Cells Treated with Lysophophatidylcholine.

OBJECTIVES: When administered soon after menopause, hormone therapy can prevent coronary heart diseases in women. To explore the mechanism underlying the cardioprotective actions of estrogen, we investigated the effects of 17ß-estradiol (17ß-E2) on the plasminogen activator system using cultured vascular smooth muscle cells (VSMCs). METHODS: VSMCs were isolated from rat aortas. Protein expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were evaluated using Western blotting and enzyme-linked immunosorbent assay, respectively. The enzyme activity of PAI-1 in a conditioned medium was assessed via reverse fibrin overlay zymography and that of t-PA was assessed via fibrin overlay zymography. Gene expression was quantified using real-time reverse transcription-polymerase chain reaction. RESULTS: Following pre-treatment for 24 hours, 17ß-E2 suppressed both protein expression and enzyme activity of PAI-1 stimulated by lysophosphatidylcholine (lysoPC) in a significant and dose-dependent manner at a near physiological concentration. Moreover, 17ß-E2 (10⁻7 M) inhibited PAI-1 gene expression, and ICI 182,780-a specific estrogen receptor antagonist-blocked the effects of 17ß-E2 on the PAI-1 protein. 17ß-E2 did not affect t-PA secretion but significantly enhanced free t-PA activity through reduced binding to PAI-1. Furthermore, 17ß-E2 suppressed intracellular reactive oxygen species production and nuclear factor-κB-mediated transcription. CONCLUSIONS: In VSMCs stimulated with lysoPC, 17ß-E2 reduced PAI-1 expression through a non-receptor-mediated mechanism via antioxidant activity as well as a receptor-mediated mechanism; however, it did not alter t-PA secretion. Of note, 17ß-E2 suppressed PAI-1 activity and concurrently enhanced t-PA activity, suggesting a beneficial influence on fibrinolysis.

Prognostic Role of Zinc Finger Homeobox 4 in Ovarian Serous Cystadenocarcinoma.

Introduction: The zinc finger homeobox 4 (ZFHX4) protein is a crucial molecular regulator of tumor-initiating stem cell-like functions. Objective: This study aimed to determine the role of ZFHX4 in the progression of ovarian serous cystadenocarcinoma (OSC). Methods: Differential gene expression ZFHX4 among low-stage (stages I and II), high-stage (stages III and IV), low-grade (grades I and II), and high-grade (grades III and IV) OSC patients was identified using four independent cohorts from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO). We compared ZFHX4 expression as a prognostic factor using Kaplan-Meier survival curves, multivariate analysis, the time-dependent area under the curve (AUC) of Uno's C-index, and the AUC of the receiver operating characteristics at 4 years post diagnosis. Results: ZFHX4 gene expression in high-stage tumors is significantly higher than in low-stage tumors (TCGA, p = 0.007; GSE9891, p = 0.001). A Kaplan-Meier analysis revealed that elevated expression of ZFHX4 was associated with a poor prognosis in OSC patients for all cohorts, regardless of stage and grade (TCGA, p = 1e-04; GSE9891, p = 0.0044; GSE13876, p = 0.00078; GSE26712, p = 0.039). Analysis of C-indices and the area under the receiver operating characteristic curve further supported this result (C-index: TCGA, 0.599; GSE9891, 0.642; GSE13876, 0.585; GSE26712, 0.597). Moreover, univariate and multivariate Cox hazards analyses confirmed the prognostic significance of ZFHX4 levels. Conclusion: Collectively, these findings suggest that ZFHX4 is a prognostic factor for OSC.