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Background and Purpose: Epigallocatechin-3-gallate (EGCG) is a component of green tea known to have chemo-preventative effects on several cancers. However, EGCG has limited clinical application, which necessitates the development of a more effective EGCG prodrug as an anticancer agent., Experimental Approach: Derivatives of EGCG were evaluated for their stability and anti-tumour activity in human chronic myeloid leukaemia (CML) K562 and KBM5 cells., Key Results: EGCG-mono-palmitate (EGCG-MP) showed most prolonged stability compared with other EGCG derivatives. EGCG-MP exerted greater cytotoxicity and apoptosis in K562 and KBM5 cells than the other EGCG derivatives. EGCG-MP induced Src-homology 2 domain-containing tyrosine phosphatase 1 (SHP-1) leading decreased oncogenic protein BCR-ABL and STAT3 phosphorylation in CML cells, compared with treatment with EGCG. Furthermore, EGCG-MP reduced phosphorylation of STAT3 and survival genes in K562 cells, compared with EGCG. Conversely, depletion of SHP-1 or application of the tyrosine phosphatase inhibitor pervanadate blocked the ability of EGCG-MP to suppress phosphorylation of BCR-ABL and STAT3, and the expression of survival genes downstream of STAT3. In addition, EGCG-MP treatment more effectively suppressed tumour growth in BALB/c athymic nude mice compared with untreated controls or EGCG treatment. Immunohistochemistry revealed increased caspase 3 and SHP-1 activity and decreased phosphorylation of BCR-ABL in the EGCG-MP-treated group relative to that in the EGCG-treated group., Conclusions and Implications: EGCG-MP induced SHP-1-mediated inhibition of BCR-ABL and STAT3 signalling in vitro and in vivo more effectively than EGCG. This derivative may be a potent chemotherapeutic agent for CML treatment., (© 2015 The British Pharmacological Society.)

Ginkgetin is a natural biflavonoid isolated from leaves of Ginkgo biloba L. Though it was known to have anti-inflammatory, anti-influenza virus, anti-fungal activity, osteoblast differentiation stimulating activity and neuro-protective effects, the underlying antitumor mechanism of ginkgetin still remains unclear. Thus, in the present study, anti-cancer mechanism of ginkgetin was elucidated in human prostate cancer PC-3 cells. Ginkgetin suppressed the viability of PC-3 cells in a concentration-dependent manner and also significantly increased the sub-G1 DNA contents of cell cycle in PC-3 cells. Ginkgetin activated caspase-3 and attenuated the expression of survival genes such as Bcl-2, Bcl-xL, survivin and Cyclin D1 at protein and mRNA levels. Consistently, pan-caspase inhibitor Z-DEVD-fmk blocked sub G1 accumulation and cleavages of PRAP and caspase 3 induced by ginkgetin in PC-3 cells. Overall, these findings suggest that ginkgetin induces apoptosis in PC-3 cells via activation of caspase 3 and inhibition of survival genes as a potent chemotherapeutic agent for prostate cancer treatment., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In this study, we investigated the role of c-Myc/ATF4/CHOP signaling pathway in sensitization of human hepatoma HepG2 cells to TRAIL. Knockdown of SIRT1 or treatment with SIRT1 inhibitor caused the up-regulation of DR5 and down-regulation of c-FLIP through modulation of c-Myc/ATF4/CHOP pathway, and subsequently enhanced the cytotoxic and apoptotic effects of TRAIL on HepG2 cells. Interestingly, SIRT1 interacted directly with c-FLIP(L) and Ku70, and treatment with SIRT1 inhibitor enhanced acetylation of Ku70 and subsequently decreased its binding to c-FLIP. And this was followed by degradation of c-FLIP. Moreover, Ku70(-/-) MEF and Ku70-knockdown HepG2 cells showed the increased levels of c-Myc, ATF4, CHOP, and DR5 and decreased level of c-FLIP. These results were followed by increased sensitivity of Ku70(-/-) MEF cells and Ku70-knockdown HepG2 cells to TRAIL compared with their control cells. These findings reveal for the first time that SIRT1 inhibition increases Ku70 acetylation, and the acetylated Ku70 with a decreased function mediates the induction of DR5 and the down-regulation of c-FLIP by up-regulating c-Myc/ATF4/CHOP pathway, and consequently promotes the TRAIL-induced apoptosis of HepG2 cells. This study provides important mechanistic insight of the synergism exhibited by SIRT1 inhibition and TRAIL., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Although brazilin [7,11b-dihydrobenz(b)indeno[1,2-d]pyran-3,6a,9,10(6H)-tetrol] isolated from Caesalpinia sappan was known to have various biological activities, including anti-inflammation, antibacteria, and antiplatelet aggregation, there is no report yet on its anticancer activity. In the present study, the anticancer mechanism of brazilin was elucidated in human multiple myeloma U266 cells. We found that brazilin significantly inhibited the activity of histone deacetylases (HDACs), transcription factors involved in the regulation of apoptosis and cell cycle arrest in U266 cells. Consistently, brazilin enhanced acetylation of histone H3 at Lys 23, indicating activation of histone acetyltransferase (HAT), and also suppressed the expressions of HDAC1 and HDAC2 at both protein and mRNA levels. Additionally, brazilin significantly increased the number of sub-G1 cell population and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells undergoing apoptosis and also activated caspase-3 and regulated the expression of Bcl-2 family proteins, including Bax, Bcl-x(L), and Bcl-2 in U266 cells, indicating that brazilin induces apoptosis through the mitochondria-dependent pathway. Interestingly, cell cycle analysis revealed that brazilin induced G2/M phase arrest along with apoptosis induction. Consistently, brazilin attenuated the expression of cyclin-dependent kinases (CDKs), such as cyclin D1, cyclin B1, and cyclin E, and also activated p21 and p27 in U266 cells. Furthermore, HAT inhibitor anacardic acid reversed activation of acetyl-histone H3 and cleavage of PARP induced by brazilin, while pan-caspase inhibitor Z-VAD-FMK001 did not affect the expression of HDAC induced by brazilin that brazilin mediates apoptosis via inactivation of HDAC in U266 cells. Notably, brazilin significantly potentiated the cytotoxic effect of standard chemotherapeutic agents, such as bortezomib or doxorubicin, in U266 cells. When our findings are taken together, they suggest that brazilin has potential as a chemotherapeutic agent alone or in combination with an anticancer agent for multiple myeloma treatment.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

It has drawn a lot of attention to target signal transducer and activator of transcription 3 (STAT3) as a potential strategy for cancer therapeutics. Using several myelogenous cell lines, the effect of genipin (an active compound of Gardenia fruit) on the STAT3 pathway and apoptosis was investigated. Genipin suppressed the constitutive STAT3 activation in U266 and U937 cells and stimulated Src homology 2 domain-containing phosphatase 1 (SHP-1), which dephosphorylates and inactivates STAT3. Specifically, genipin blocked STAT3 activation via repressing the activation of c-Src, but not Janus kinase 1 (JAK1). Genipin also downregulated the expression of STAT3 target genes including Bcl-2, Bcl-x(L) , Survivin, Cyclin D1, and VEGF. Conversely, protein tyrosine phosphatase inhibitor pervanadate blocked genipin induced STAT3 inactivation. Using DNA fragmentation or TUNEL assays, we demonstrated the apoptotic effect of genipin on U266, MM.1S, and U937 cells. Furthermore, genipin effectively potentiated the cytotoxic effect of chemotherapeutic agents, such as bortezomib, thalidomide, and paclitaxel in U266 cells. Our data suggest that through regulation of Src and SHP-1, genipin antagonizes STAT3 for the induction of apoptosis in myeloma cells., (Copyright © 2011 Wiley-Liss, Inc.)

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.

Aim of the Study: The inhibitory effect of Dryopteris crassirhizoma on the proliferation of human metastatic prostate PC3-MM2 cells and the mechanism of action were examined to identify its anti-cancer properties. The effect of the extract on cell cycle progression and its combined cytotoxic effect with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on PC3-MM2 cells were also investigated., Materials and Methods: The anti-proliferative effects of Dryopteris crassirhizoma were examined by culturing PC3-MM2 cells in the presence or absence of various concentrations of Dryopteris crassirhizoma extract, and the inhibitory effects on cell proliferation were determined by Cell Counting Kit (CCK)-8 analysis. The quantities of apoptosis-inducing proteins were measured by western blotting analysis. Cell cycle progression was analyzed by PI staining using flow cytometry., Results: Dryopteris crassirhizoma (50 and 100 microg/ml) inhibited markedly the proliferation of PC-3 and PC3-MM2 cells without cytotoxicity to normal (spleen) cells from BALB/C mice. Dryopteris crassirhizoma (100 microg/ml) effectively induced apoptosis through the activation of caspase-3, -8, -9, bid, and PARP in PC3-MM2 cells. The cells exposed to Dryopteris crassirhizoma increased significantly the accumulation of the DNA contents in the G0/G1 phase and sub-G1 phase in contrast to the control. The combined cytotoxic effects of Dryopteris crassirhizoma and TRAIL induced the increased activity of 29% in contrast to the sum of the inhibitory effects of each agent alone., Conclusions: Dryopteris crassirhizoma has anti-cancer properties by inducing cell cycle arrest and apoptosis through the extrinsic and intrinsic pathway in PC3-MM2 cells. The extract also showed a combined effect with TRAIL on the inhibition of proliferation in the cells. These findings suggest that possibly its extract could be used for treating androgen-independent prostate cancer with minimal side effects., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

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.

Icariside II (IS) isolated from the roots of Epimedium koreanum Nakai was known to have antioxidant activity and inhibit melanogenesis and hypoxia inducible factor. We report here for the first time that IS induces apoptosis through its anti-inflammatory effects in PC-3 prostate cancer cells. IS exerted cytotoxicity against PC-3 cells with IC(50) of approximately 20 microM. IS suppressed both constitutive and arachidonic acid (AA)-induced cyclooxygenase-2 (COX-2) expression as well as reduced prostaglandin E2 (PGE2) levels in PC-3 cells even at a low concentrations (5 and 10 microM). Additionally, IS increased sub G1 apoptotic portion and exhibited terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)-positive apoptotic bodies in PC-3 cells at higher concentrations (20 and 40 microM). Furthermore, IS attenuated the mitochondrial membrane potential, released cytochrome C into cytosol, activated caspase-9, -8, and -3 expressions and cleaved poly (ADP-ribose) polymerase (PARP) in PC-3 cells. Consistently, COX-2, inducible NO synthase (iNOS), and vascular endothelial growth factor (VEGF) expressions were suppressed while in parallel inducing apoptosis in hormone-independent prostate carcinoma cells PC-3. Moreover, exogeneous PGE2 inhibited IS induced PARP cleavage in PC-3 cells and also knockdown of COX-2 by siRNA potentiated IS induced PARP cleavage, thereby implicating the critical role of COX-2 pathway in IS induced apoptosis. Taken together, these findings demonstrate that IS initiates the inhibition of COX-2/PGE(2) pathway and then induces apoptosis mainly via mitochondrial dependent pathway in PC-3 prostate cancer cells as a potent cancer chemotherapeutic agent.

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-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.

Aim of This Study: Korean red ginseng (KRG, Panax ginseng C.A. Meyer Radix rubra) has been used to treat various diseases including cancer. However, the molecular mechanisms responsible for KRG extract induced apoptosis and telomerase inhibition remain unclear., Materials and Methods: The hot water extract from KRG was used to evaluate the mechanism of induction of apoptosis in U937 human leukemia cells and its effects on cyclooxgenase-2 (COX-2) and telomerase activity., Results: KRG extract treatment to U937 cells resulted in growth inhibition and induction of apoptosis in a concentration-dependent manner as measured by hemacytometer counts, MTT assay, fluorescence microscopy, agarose gel electrophoresis and flow cytometry analysis. The increase in apoptosis was associated with the down-regulation of antiapoptotic Bcl-2, Bcl-X(L), and IAPs family members, and the activation of caspase-3. KRG extract treatment also decreased the expression levels of COX-2 and inducible nitric oxide synthase. Furthermore, KRG extract treatment progressively down-regulated the expression of human telomerase reverse transcriptase, a main determinant of the telomerase enzymatic activity, with inhibiting the expression of c-Myc in a concentration-dependent manner., Conclusions: These results provide important new insights into the possible molecular mechanisms of the anticancer activity of KRG extract.

Focal adhesion kinase (FAK) is a signaling molecule associated with cell survival. Previously, we showed that thimerosal, a reactive oxygen species (ROS) generator, can acutely induce FAK tyrosine phosphorylation (within minutes) and chronically induce apoptosis (within days) by redox modulation in HeLa S cells. In the present study, we report that a prolonged oxidative stress by thimerosal induces a remarkable cleavage of FAK, which is accompanied with apoptosis. In fact, the kinetics of FAK cleavage has a good correlation with and actually preceding the apoptosis that was independent of anoikis. The effects were almost completely blocked by the pretreatment with either N-acetyl-l-cysteine (ROS scavenger) or Z-VAD-FMK (pan-caspase inhibitor), suggesting ROS-induced caspase activation as a key mechanism. They could be also reproduced by hydrogen peroxide alone, which appeared to be responsible for thimerosal-mediated oxidative stress-induced apoptosis. Additionally, the down regulation of FAK with antisense oligonucleotide dramatically augmented thimerosal-induced apoptosis. We could observe similar results using human corneal epithelial cells. Taken together, our results show that FAK is a critical cellular target of caspases during oxidative stress (particularly by hydrogen peroxide), resulting in the acceleration of subsequent apoptosis regardless of the anchorage status of cells. From the present results, it is more likely that not cell detachment but the proteolytic cleavage (or inhibition) of FAK is a key modulator as well as a promising indicator of apoptosis in epithelial cells under oxidative stress.

Polychlorinated biphenyls (PCBs), a group of persistent and widespread environmental pollutants, are considered to be immunotoxic, carcinogenic, and to induce apoptosis. However, the cellular mechanisms underlying the action of PCBs have not been established. Here, we investigated the effects of PCBs on the induction of cyclooxygenase-2 (COX-2). Among the several congeners examined, only 2,2',4,6,6'-pentachlorobiphenyl (PeCB) specifically increased the COX-2 promoter activity, and the levels of COX-2 mRNA and protein, and thereby enhanced prostaglandin E2 (PGE2) synthesis in Rat-1 cells. By conducting mutation analyses of the COX-2 promoter and its transcription factor, we found that the CRE site in COX-2 promoter and c-Jun are important for increased COX-2 promoter activity induced by 2,2',4,6,6'-PeCB. In addition, 2,2',4,6,6'-PeCB-stimulated COX-2 induction was reduced by the specific MAPK kinase (MEK) inhibitor, PD98059, and in p53-deficient cells, implying that COX-2 induction requires the activation of ERK1/2 MAPK and p53. The selective COX-2 inhibitor, NS-398, potentiated the 2,2',4,6,6'-PeCB-induced mitochondrial apoptotic pathway involved in Bcl-xL attenuation, cytochrome c release and the subsequent activation of caspase-3. Furthermore, the cell death was prevented by PGE2 treatment, suggesting that 2,2',4,6,6'-PeCB-induced apoptosis is restricted by prostaglandin upregulation by COX-2. Taken together, these results demonstrate that 2,2',4,6,6'-PeCB-induced COX-2 expression may be an important compensatory mechanism for abating 2,2',4,6,6'-PeCB toxicity.

The failure to treat metastatic cancer with multidrug resistance is a major problem for successful cancer therapy, and the molecular basis for the association of metastatic phenotype with resistance to therapy is still unclear. In this study, we revealed that various metastatic cancer cells showed consistently higher levels of antiapoptotic proteins, including Bcl-2, nuclear factor-kappaB, MDM2, DNA-dependent protein kinase (DNA-PK), and epidermal growth factor receptor (EGFR), and lower levels of proapoptotic proteins, including Bax and p53 than low metastatic parental cells. This was followed by chemo- and radioresistance in metastatic cancer cells compared with their parental cells. EGFR and DNA-PK activity, which are known to be associated with chemo- and radioresistance, were demonstrated to be mutually regulated by each other. Treatment with PKI166, an EGFR inhibitor, suppressed etoposide-induced activation of DNA-PK in A375SM metastatic melanoma cells. In addition, PKI166 enhanced markedly the chemosensitivities of metastatic cancer cell sublines to various anticancer drugs in comparison with those of low metastatic cancer cells. These results suggest that the activities of DNA-PK and EGFR, which is positively correlated with each other, may contribute to metastatic phenotype as well as therapy resistance, and the EGFR inhibitor enhances the effect of anticancer drugs against therapy-resistant metastatic cancer cells via suppression of stress responses, including activation of DNA-PK.

The neurotoxicity of dithiocarbamates has been previously reported, however, the detailed mechanism underlying the neurotoxicity is still not fully understood. Among the dithiocarbamates, we investigated thiram and ziram in a neuronal-like pheochromocytoma (PC12) cells. Thiram and ziram strongly induced cell death in both dose- and time-dependent manners with the LC(50) of 0.3 and 2 microM, respectively. The cell death showed typical apoptotic features, such as DNA fragmentation and an increase of subdiploidy nuclei. Interestingly, both thiram and ziram induced rapid and sustained increases of intracellular Ca(2+) in PC12 cells, which were almost completely blocked by flufenamic acid (FFA), an inhibitor of non-selective cation channel. BAPTA-AM, an intracellular Ca(2+) chelator, inhibited the thiram- and ziram-induced apoptotic cell death. These results suggest that thiram and ziram induce apoptotic neuronal cell death by Ca(2+) influx through non-selective cation channels. The present study may provide a clue for understanding the mechanism of neurotoxicity of thiram and ziram.

Abstract: Although the flavonoid icariside II exhibits anti-inflammatory and anti-cancer activities, its molecular targets/pathways in human multiple myeloma cells are poorly understood. To analyze the effects on signal transducer and activator of transcription 3 (STAT3) signaling and apoptosis, U266 multiple myeloma cells were treated with icariside II and performed Western blotting, electrophoretic mobility gel shift assay (EMSA), RT-PCR, proliferation assay, cell cycle analysis and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Icariside II inhibited STAT3 activation and enhanced the expression of SHP-1 and PTEN through inhibiting Janus activated kinase 2 (JAK2) and c-Src. Icariside II down-regulated the expression of STAT3 target genes Bcl-2, Bcl-xL, survivin, cyclin D1, COX-2 and vascular endothelial growth factor (VEGF). Also, icariside II enhanced poly (ADP-ribose) polymerase (PARP) cleavage and caspase-3 activation. Pervanadate reversed the icariside II-mediated STAT3 inactivation and also blocked the cleavages of caspase-3 and PARP, suggesting involvement of STAT3 pathway in icariside II-induced apoptosis. Furthermore, icariside II enhanced the apoptotic effects of clinically used drugs thalidomide and bortezomib in U266 cells. Icariside II could be a potential therapeutic intervention agent alone or in combination with current drugs for multiple myeloma as a novel blocker of STAT3 signaling cascades at multiple levels, contributing to its anti-proliferative and anti-apoptosis. [ABSTRACT FROM AUTHOR]

3′-Phosphoinositide-dependent kinase-1 (PDK1) has been identified for its ability to phosphorylate and activate Akt. Accumulated studies have shown that the activation of the PDK1/Akt pathway plays a pivotal role in cell survival, proliferation, and tumorigenesis. Therefore, the PDK1/Akt pathway is believed to be a critical target for cancer intervention. In this paper, we report the discovery of a new function of phenothiazines, widely known as antipsychotics, inhibiting PDK1/Akt pathway. Upon epidermal growth factor (EGF) stimulation, phenothiazines specifically suppressed the kinase activity of PDK1 and the phosphorylation level of Akt. The inhibition of PDK1/Akt kinase resulted in suppression of EGF-induced cell growth and induction of apoptosis in human ovary cancer cells. In particular, phenothiazines were highly selective for downstream targets of PDK1/Akt and did not inhibit the activation of phosphatidylinositol 3-kinase (PI3K), EGFR, or extracellular signal-regulated kinase 1/2 (ERK1/2). In particular, phenothiazines effectively suppressed tumor growth in nude mice of human cancer cells. Taken together, these findings provide strong evidence for novel function of phenothiazines, pharmacologically targeting PDK1/Akt for anticancer drug discovery. [ABSTRACT FROM AUTHOR]

Abstract: We investigated the molecular mechanisms responsible for fisetin-induced apoptosis in U266 cells. Fisetin elicited the cytotoxicity in U266 cells, manifested as an increased fraction of the cells with sub-G1 content or stained positively with TUNEL labeling. Fisetin enhanced caspase-3 activation, downregulation of Bcl-2 and Mcl-1L, and upregulation of Bax, Bim and Bad. Fisetin activated AMPK as well as its substrate acetyl-CoA carboxylase (ACC), along with a decreased phosphorylation of AKT and mTOR. Fisetin also stimulated generation of ROS in U266 cells. Conversely, compound C or N-acetyl-l-cystein blocked fisetin-induced apoptosis. Our data suggest that fisetin-induced apoptosis in U266 cells is through ROS and AMPK pathways. [Copyright &y& Elsevier]

SAMHD1 limits HIV-1 infection in non-dividing myeloid cells by decreasing intracellular dNTP pools. HIV-1 restriction by SAMHD1 in these cells likely prevents activation of antiviral immune responses and modulates viral pathogenesis, thus highlighting a critical role of SAMHD1 in HIV-1 physiopathology. Here, we explored the function of SAMHD1 in regulating cell proliferation, cell cycle progression and apoptosis in monocytic THP-1 cells. Using the CRISPR/Cas9 technology, we generated THP-1 cells with stable SAMHD1 knockout. We found that silencing of SAMHD1 in cycling cells stimulates cell proliferation, redistributes cell cycle population in the G 1 /G 0 phase and reduces apoptosis. These alterations correlated with increased dNTP levels and more efficient HIV-1 infection in dividing SAMHD1 knockout cells relative to control. Our results suggest that SAMHD1, through its dNTPase activity, affects cell proliferation, cell cycle distribution and apoptosis, and emphasize a key role of SAMHD1 in the interplay between cell cycle regulation and HIV-1 infection. [ABSTRACT FROM AUTHOR]

Abstract: Icariside II (IS) isolated from the roots of Epimedium koreanum Nakai was known to have antioxidant activity and inhibit melanogenesis and hypoxia inducible factor. We report here for the first time that IS induces apoptosis through its anti-inflammatory effects in PC-3 prostate cancer cells. IS exerted cytotoxicity against PC-3 cells with IC50 of approximately 20μM. IS suppressed both constitutive and arachidonic acid (AA)-induced cyclooxygenase-2 (COX-2) expression as well as reduced prostaglandin E2 (PGE2) levels in PC-3 cells even at a low concentrations (5 and 10μM). Additionally, IS increased sub G1 apoptotic portion and exhibited terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)-positive apoptotic bodies in PC-3 cells at higher concentrations (20 and 40μM). Furthermore, IS attenuated the mitochondrial membrane potential, released cytochrome C into cytosol, activated caspase-9, -8, and -3 expressions and cleaved poly (ADP-ribose) polymerase (PARP) in PC-3 cells. Consistently, COX-2, inducible NO synthase (iNOS), and vascular endothelial growth factor (VEGF) expressions were suppressed while in parallel inducing apoptosis in hormone-independent prostate carcinoma cells PC-3. Moreover, exogeneous PGE2 inhibited IS induced PARP cleavage in PC-3 cells and also knockdown of COX-2 by siRNA potentiated IS induced PARP cleavage, thereby implicating the critical role of COX-2 pathway in IS induced apoptosis. Taken together, these findings demonstrate that IS initiates the inhibition of COX-2/PGE2 pathway and then induces apoptosis mainly via mitochondrial dependent pathway in PC-3 prostate cancer cells as a potent cancer chemotherapeutic agent. [Copyright &y& Elsevier]

Abstract: 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-κB pathway. In addition, apicidin decreased the level of NF-κB-dependent Bcl-xL, 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. [Copyright &y& Elsevier]

Hwaeumjeon is a classical prescription that has been traditionally used for treatment of urogenital diseases with no scientific evidences until now. Thus, the present study was performed to evaluate antitumor mechanism of ethanolic Hwaeumjeon (EHEJ). 2-Dimensional electrophoresis (2-DE) proteomic analysis, cell culture study, and Western blotting on apoptosis and prostate-specific antigen (PSA) related proteins were carried out in LNCaP prostate cancer cells. Eight spots with significant increased or decreased expression revealed by 2-DE based comparative proteomic analysis were identified as an increased protein ENC-1AS, four decreased proteins such as RAB34, SFRS1, heat shock 27, and proteasome activator, and three novel proteins such as Rho GDP dissociation inhibitor alpha, cytoplasmic antiproteinase, and EIF3EIP protein in EHEJ-treated LNCaP cells. In addition, EHEJ selectively inhibited the growth of LNCaP prostate cancer cells compared to normal human umbilical vein endothelial cells. Furthermore, EHEJ inhibited PSA and androgen receptor (AR) expression in androgen sensitive LNCaP prostate cancer cells at nontoxic concentrations. Also, EHEJ increased sub-G1 apoptotic portion, activated caspase-9 and -3, cleaved poly (ADP-ribose) polymerase (PARP) and increased the ratio of Bax to Bcl-2. Interestingly, EHEJ also attenuated phosphatidylinositol-3 kinase (PI3K) expression and suppressed the phosphorylation of survival gene AKT, ERK, and HSP27 in LNCaP cells. Consistently, PI3K and ERK inhibitors potentiated EHEJ-induced cytotoxicity and overexpression of Bcl-2 attenuated EHEJ-mediated apoptosis in LNCaP cells. These findings suggest that EHEJ induces mitochondrial dependent apoptosis partly via PI3K/AKT/HSP27/ERK pathways and inhibits PSA and AR in LNCaP cells as a prostate cancer chemopreventive candidate. [Copyright &y& Elsevier]

Abstract: 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-FLIPL 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-FLIPL 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-FLIPL expression and co-treatment with TRAIL further reduced the level of c-FLIPL, 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-FLIPL 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. [Copyright &y& Elsevier]

Since DNA-dependent protein kinase (DNA-PK) has been known to play a protective role against drug-induced apoptosis, the role of DNA-PK in the regulation of mitochondrial heat shock proteins by anticancer drugs was examined. The levels of basal and drug-induced mitochondrial heat shock proteins of drug-sensitive parental cells were higher than those of multidrug-resistant (MDR) cells. We also demonstrated that the development of MDR might be correlated with the increased expression of Ku-subunit of DNA-PK and concurrent down-regulation of mitochondrial heat shock proteins. The basal mtHsp70 and Hsp60 levels of Ku70−/− cells, which were known to be sensitive to anticancer drugs, were higher than those of parental MEF cells, but conversely these mitochondrial heat shock proteins of R7080-6 cells over-expressing both Ku70 and Ku80 were lower than those of parental Rat-1 cells. Also, the mtHsp70 and Hsp60 levels of DNA-PKcs-deficient SCID cells were higher than those of parental CB-17 cells. Our results suggest the possibility that mitochondrial heat shock protein may be one of determinants of drug sensitivity and could be regulated by DNA-PK activity. [Copyright &y& Elsevier]

Polychlorinated biphenyls (PCBs) are a group of persistent and widespread environmental pollutants, and known to affect signaling molecules. Phospholipase A2 (PLA2) mediates cellular destructive processes as well as normal physiological responses in neuronal cells. In this study, we examined whether PLA2 can be activated by PCBs in PC12 cells. Of the congeners tested, ortho-substituted PCBs were found to induce PLA2 activation. PLA2 activation by 2,2′,4,6-tetrachlorobiphenyl (TeCB), the most potent congener, was inhibited by bromoenol lactone (BEL), a calcium-independent PLA2 (iPLA2) inhibitor, and methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 and iPLA2 inhibitor. In the case of Ca2+, although 2,2′,4,6-TeCB increased [Ca2+]i in the presence of extracellular Ca2+, PLA2 activation was not inhibited by EGTA and 1,2-bis (o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra (acetoxy-methyl) ester, an extracellular and an intracellular Ca2+ chelator, respectively. On the other hand, 2,2′,4,6-TeCB-induced Ca2+ increase was partially inhibited by BEL and MAFP. In addition, 2,2′,4,6-TeCB induced apoptotic cell death in these cells. Taken together, our results suggest that ortho-substituted PCBs might induce apoptosis through PLA2-mediated Ca2+ influx, which provides a clue to understand the mechanism of neurotoxic effects of ortho-substituted PCBs. [Copyright &y& Elsevier]