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Previous studies have indicated that the sensitivity of breast cancer cells to tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL)‑induced apoptosis is associated with the expression of death receptors on the cell membrane. However, drug resistance limits the use of TRAIL in cancer therapy. Numerous studies have indicated that death receptors, which induce apoptosis, are upregulated by the endoplasmic reticulum (ER) stress response. 3‑Bromopyruvate (3‑BP), an anticancer agent, inhibits cell growth and induces apoptosis through interfering with glycolysis. In the present study, it was demonstrated that 3‑BP synergistically sensitized breast cancer cells to TRAIL‑induced apoptosis via the upregulation of death receptor 5 (DR5). Furthermore, we found that the protein levels of glucose‑related protein 78 (GRP78) and CCAAT‑enhancer‑binding protein homologous protein (CHOP) increased following treatment with 3‑BP. The expression of Bax (in MCF‑7 cells) and caspase‑3 (in MDA‑MB‑231 cells) increased following co‑treatment with 3‑BP and TRAIL, whereas the expression of the anti‑apoptotic protein Bcl‑2 decreased. In order to investigate the molecular mechanism regulating this effect, the expression of adenosine monophosphate‑activated protein kinase (AMPK), activated by 3‑BP, was determined. It was demonstrated that phosphorylated‑AMPK was upregulated following treatment with 3‑BP. Notably, Compound C, an AMPK inhibitor, reversed the effects of 3‑BP. Finally, a synergistic antitumor effect of 3‑BP and TRAIL was observed in MCF‑7 cell xenografts in nude mice. In conclusion, these results indicated that 3‑BP sensitized breast cancer cells to TRAIL via the AMPK‑mediated upregulation of DR5.

TRAIL‑R2 (DR5), one of the death receptors, can activate the extrinsic apoptosis pathway, while cellular FLICE‑inhibitory protein (c‑FLIP) can inhibit this pathway. Both of them play important roles in the occurrence and development of most tumors. To date, there is no relevant report concerning the relationship between expression of DR5 and c‑FLIP protein and clinicopathological/prognostic implications in patients with non‑small cell lung cancer (NSCLC) treated with surgical resection and chemotherapy. Thus, the aim of the present study was to investigate the potential prognostic significance of DR5 and c‑FLIP in NSCLC patients and their predictive roles in the chemotherapeutic response. In the present study, DR5 and c‑FLIP were detected by immunohistochemistry (IHC) in tissue microarrays of NSCLC. The results showed that the expression levels of DR5 and c‑FLIP were significantly higher in lung squamous cell carcinoma (SCC) and lung adenocarcinoma (ADC) tissues compared with levels noted in the non‑cancerous control lung tissues (all P

Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) is a cytokine with the potential to induce cancer cell‑specific apoptosis with minimal toxicity to normal cells. Therefore, the resistance of certain cancer cells to TRAIL is a major concern and agents that can either enhance TRAIL capabilities or overcome TRAIL resistance are necessary for the development of cancer treatments. The present study investigated whether the antidepressant drug amitriptyline could sensitize TRAIL‑resistant A549 lung cancer cells and enhance TRAIL‑induced apoptosis. Antidepressants are usually prescribed to cancer patients to relieve emotional distress, such as depression or dysthymia. The present study revealed for the first time, to the best of our knowledge, that amitriptyline increased death receptor (DR) 4 and 5 expression, a requirement for TRAIL‑induced cell death. Genetic inhibitors of DR4 and DR5 significantly reduced amitriptyline‑enhanced TRAIL‑mediated apoptosis. Additionally, the present study explored whether blocking autophagy increased DR4 and DR5 expression. Blocking autophagy flux with the final stage autophagy inhibitor chloroquine (CQ) also upregulated DR4 and DR5 expression. TRAIL in combination with amitriptyline or CQ significantly increased the expression of apoptosis‑indicator proteins cleaved caspase‑8 and caspase‑3. The expression levels of LC3‑II and p62 were significantly higher in amitriptyline‑treated cells, which confirmed that amitriptyline blocks autophagy by inhibiting the fusion of autophagosomes with lysosomes. Overall, the present results contributed to understanding the mechanism responsible for the synergistic anticancer effect of amitriptyline and TRAIL and also presented a novel mechanism involved in DR4 and DR5 upregulation.

Tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL) selectively induces apoptosis in cancer cells, with minimal toxicity to normal tissues. However, accumulating evidence suggests that certain cancer types are insensitive to TRAIL signaling. The aim of this study was to identify an effective combination regimen, which can overcome TRAIL resistance in renal cancer cell. Herein, we found that human renal carcinoma cells (RCCs) are widely resistant to TRAIL‑mediated growth inhibition and subsequently identified that andrographolide (Andro), a major constituent of Andrographis paniculate, an annual herbaceous plant in the family Acanthaceae, counteracts TRAIL resistance in RCCs. Combined treatment with TRAIL and Andro suppressed cell viability as determined by MTS and proliferation as determined by EdU in a dose‑dependent manner and inactivated the clonogenic and migration ability of RCCs. Andro significantly enhances TRAIL‑mediated cell cycle arrest at the G2/M phase as determined by flow cytometry and senescence. Moreover, Andro restored TRAIL signaling, which in turns activated pro‑apoptosis caspases as determined by immunoblot assay. The TRAIL receptor, death receptor (DR)4, but not DR5, was found to be significantly upregulated in Andro‑treated RCC cells, which contributed to the role of Andro as a TRAIL sensitizer. The present study demonstrated that the combined treatment of Andro and TRAIL has potential therapeutic value against renal cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in cancer cells by binding to its receptors, death receptor 4 (DR4) and DR5, without affecting normal cells, and is therefore considered to be a promising antitumor agent for use in cancer treatment. However, several studies have indicated that most glioma cell lines display resistance to TRAIL-induced apoptosis. To overcome such resistance and to improve the efficacy of TRAIL-based therapies, identification of ideal agents for combinational treatment is important for achieving rational clinical treatment in glioblastoma patients. The main aim of this study was to investigate whether interferon-β (IFN-β) (with its pleiotropic antitumor activities) could sensitize malignant glioma cells to TRAIL-induced apoptosis using glioma cell lines. TRAIL exhibited a dose-dependent antitumor effect in all of the 7 types of malignant glioma cell lines, although the intensity of the effect varied among the cell lines. In addition, combined treatment with TRAIL (low clinical dose: 1 ng/ml) and IFN-β (clinically relevant concentration: 10 IU/ml) in A-172, AM-38, T98G, U-138MG and U-251MG demonstrated a more marked antitumor effect than TRAIL alone. Furthermore, the antitumor effect of the combined treatment with TRAIL and IFN-β may be enhanced via an extrinsic apoptotic system, and upregulation of DR5 was revealed to play an important role in this process in U-138MG cells. These findings provide an experimental basis to suggest that combined treatment with TRAIL and IFN-β may offer a new therapeutic strategy for malignant gliomas.

Myelodysplastic syndromes (MDSs) are a group of malignant clone hematopoietic stem-cell diseases, and the evolution and progression of MDS depend on the abnormal apoptosis of bone marrow cells. Our previous studies have indicated that sperm-associated antigen 6 (SPAG6), located in the uniparental disomy regions of myeloid cells, is overexpressed in patients with MDS as compared to controls, and SPAG6 can inhibit apoptosis of SKM-1. However, the concrete mechanism is still unclear. In the present study, it was found that the TNF-related apoptosis-inducing ligand (TRAIL)signal pathway was activated when the expression of SPAG6 was inhibited by SPAG6-shRNA lentivirus in SKM-1 cells. Additionally, the results of flow cytometry, Cell Counting Kit-8 assay and western blot analysis implied that the TRAIL signal pathway could be inhibited by a high expression of SPAG6. However, SPAG6 cannot influence the expression of TRAIL death receptors, except for FADD. Additionally the interaction between FADD and TRAIL death receptors also increased in SKM-1 cells infected with SPAG6-shRNA lentivirus. Thus, our study demonstrates that SPAG6 may regulate apoptosis in SKM-1 through the TRAIL signal pathway, indicating that SPAG6 could be a potential therapeutic target.

Heat-shock protein (Hsp) 70, known as a pro-survival protein, is aberrantly expressed in several malignancies. The small molecule 2-phenylethyenesulfonamide (PES), also referred to as pifithrin-μ, is known as an HSP70 inhibitor, which exhibits antitumor activities in a variety of cancer cell lines. However, little is known about its effect on non-small cell lung cancer (NSCLC) cell lines. This study aimed to investigate the effect of PES on human NSCLC cell lines A549 and H460, and explore the possible underlying mechanism of action. Cell viability assay by using CCK-8 kits was performed to demonstrate that PES dose- and time-dependently inhibited proliferation of A549 and H460 cells. Wound healing assay and Transwell migration assay results indicated that PES inhibited cell migration of A549 and H460 cells. Flow cytometry results demonstrated that PES resulted in G0/G1 phase cell cycle arrest, and induced apoptosis via a caspase-dependent manner in A549 and H460 cells. Western blotting results suggested that phosphorylation of AKT and ERK was inhibited by PES treatment. In addition, death receptor 4 (DR4) and DR5 were increased by PES treatment. Overexpression of Hsp70 in A549 cells attenuated the growth inhibitory efficiency of PES. Knockdown of Hsp70 in A549 cells enhanced sensitivity of PES to cell growth inhibition, suggesting that the inhibitory effect of PES on cell proliferation is specifically through Hsp70-dependent mechanism. PES and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts a potent synergistic effect on cell proliferation inhibition and induction of apoptosis in A549 and H460 cells. In a mouse xenograft model of lung cancer by A549 cells, PES treatment displayed significant inhibitory effects on tumor growth. All these findings suggest that PES shows antitumor activity against human NSCLC in vitro and in vivo, and therefore may be a promising agent for use to the treatment of NSCLC.

Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response 1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0 value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0 value of pc-H/E‑hsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.

Manumycin A (Manu A) is a natural product isolated from Streptomyces parvulus and has been reported to have anti-carcinogenic and anti-biotic properties. However, neither its molecular mechanism nor its molecular targets are well understood. Thus, the aim of the present study was to explore the possibility that Manu A has cancer preventive and chemotherapeutic effects on malignant pleural mesothelioma (MPM) through regulation of Sp1 and induction of mitochondrial cell death pathway. Manu A inhibited the cell viability of MSTO-211H and H28 cells in a concentration‑dependent manner as determined by MTS assay. IC50 values were calculated as 8.3 and 4.3 µM in the MSTO-311H and H28 cells following 48 h incubation, respectively. Manu A induced a significant increase in apoptotic indices as shown by DAPI staining, Annexin V assay, multi-caspase activity and mitochondrial membrane potential assay. The downregulation of Sp1 mRNA and protein expression by Manu A led to apoptosis by suppressing Sp1-regulated proteins (cyclin D1, Mcl-1 and survivin). Manu A decreased the protein levels of BID, Bcl-xL and PARP while it increased Bax levels. Manu A caused depolarization of the mitochondrial membrane with induction of CHOP, DR4 and DR5. Our results demonstrated that Manu A exerted anticancer effects by inducing apoptosis via inhibition of the Sp1-related signaling pathway in human MPM.

Drug-resistant neuroblastoma remains a major challenge in paediatric oncology and novel and less toxic therapeutic approaches are urgently needed to improve survival and reduce the side effects of traditional therapeutic interventions. Mesenchymal stem cells (MSCs) are an attractive candidate for cell and gene therapy since they are recruited by and able to infiltrate tumours. This feature has been exploited by creating genetically modified MSCs that are able to combat cancer by delivering therapeutic molecules. Whether neuroblastomas attract systemically delivered MSCs is still controversial. We investigated whether MSCs engineered to express tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) could: i) cause death of classic and primary neuroblastoma cell lines in vitro; ii) migrate to tumour sites in vivo; and iii) reduce neuroblastoma growth in xenotransplantation experiments. We observed that classic and primary neuroblastoma cell lines expressing death receptors could be killed by TRAIL-loaded MSCs in vitro. When injected in the peritoneum of neuroblastoma-bearing mice, TRAIL-MSCs migrated to tumour sites, but were unable to change the course of cancer development. These results indicated that MSCs have the potential to be used to deliver drugs in neuroblastoma patients, but more effective biopharmaceuticals should be used instead of TRAIL.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent anticancer agent possessing the ability to induce apoptosis in various cancer cells but not in non‑malignant cells. However, certain type of cancer cells are resistant to TRAIL‑induced apoptosis and some acquire resistance after the first treatment. So development of an agent that can reduce or avoid resistance in TRAIL‑induced apoptosis has garnered significant attention. The present study evaluated the anticancer potential of hispolon in TRAIL‑induced apoptosis and indicated hispolon can sensitize cancer cells to TRAIL. As the mechanism of action was examined, hispolon was found to activate caspase‑3, caspase‑8 and caspase‑9, while downregulating the expression of cell survival proteins such as cFLIP, Bcl‑2 and Bcl‑xL and upregulating the expression of Bax and truncated Bid. We also found hispolon induced death receptors in a non‑cell type‑specific manner. Upregulation of death receptors by hispolon was found to be p53-independent but linked to the induction of CAAT enhancer binding protein homologous protein (CHOP). Overall, hispolon was demonstrated to potentiate the apoptotic effects of TRAIL through downregulation of anti‑apoptotic proteins and upregulation of death receptors linked with CHOP and pERK elevation.

Cells from dental tissues have a mesenchymal stem cell (MSC) phenotype, are multipotent and can differentiate into osteoblastic cells, as we have previously found. MSCs, due to their tumor‑homing ability, are currently being used as cell‑based delivery systems for cancer protein therapeutics, such as the TNF‑related apoptosis‑inducing ligand (TRAIL). In the present study we revealed that dental pulp stem cells (DPSCs) expressed TRAIL to a greater extent when they were differentiated into the osteoblastic lineage. TRAIL affected the viability of undifferentiated DPSCs, while osteoblastic differentiated DPSCs were not sensitive to TRAIL. The expression trend of TRAIL receptors underwent changes during the osteoblastic differentiation of DPSCs exhibiting low DcR2 and high DR5 levels in the undifferentiated DPSCs and an opposite scenario was presented in the differentiated cells. The sensitivity of the undifferentiated DPSCs to the TRAIL‑apoptotic effect was also associated with low levels of intracellular anti‑apoptotic proteins, such as c‑FLIP, XIAP and the activation of caspase‑8 and ‑3. DPSC‑differentiated osteoblasts expressing high TRAIL levels were capable to affect the cell viability of the human myeloma cell line H929, thus representing an effective anticancer therapeutic method.

Ovarian cancer is the leading cause of cancer‑ associated mortality in the female reproductive system. Interleukin (IL)‑33 and its receptor IL 1 receptor like 1 (also termed ST2) are expressed by many cell types including epithelial cells. The role of IL‑33 in the pathogenesis of neoplasia remains controversial. The authors previously demonstrated that IL‑33 inhibits the growth of pancreatic cancer cells. The present study was performed to explore if IL‑33 has any direct effects on ovarian cancer cells. A clonogenic survival assay, immunohistochemistry (IHC), proliferation kit and caspase‑3 activity kit were all used to evaluate the direct effects of IL‑33 on cell proliferation and apoptosis of a widely studied ovarian cancer cell line, A2780. The possible molecular mechanisms were further evaluated with reverse transcription‑polymerase chain reaction and IHC. It was demonstrated that the percentage of colonies and the optical density value of cancer cells were all increased in the presence of IL‑33; however, the relative caspase‑3 activity in cancer cells was decreased in the presence of IL‑33. Molecular mechanism studies revealed that the pro‑proliferative effect of IL‑33 on cancer cells was associated with decreased levels of p27, and the anti‑apoptotic effect of IL‑33 was associated with levels of Fas cell surface death receptor (Fas) and tumor necrosis factor‑related apoptosis‑inducing ligand receptor 1 (TRAILR1). Therefore, IL‑33 promoted proliferation and inhibited apoptosis of ovarian cancer cells by downregulation of p27, Fas and TRAILR1. Contrary to previous studies demonstrating an anti‑tumor effort in pancreatic cancer, the results of the present study indicated that IL‑33 exhibited a significant onco‑promoting effect on ovarian cancer. Accordingly, the inhibition of IL‑33 may be a promising therapeutic strategy for ovarian cancer.

The aim of the present study was to explore the effect of 6-fluoro-2-(3-fluorophenyl)-4-(cyanoanilino) quinazoline (HMJ-30) on the anti-angiogenic properties and apoptosis-related mechanism of human umbilical vein endothelial cells (HUVECs). In this study, HMJ-30 dose- and time-dependently inhibited the viability of HUVECs. We also found that HMJ-30 enhanced disruption of tube-like structures and suppressed cell migration in HUVECs after vascular endothelial growth factor (VEGF) induction. HMJ-30 was also observed to inhibit vessel branching and sprouting in chicken chorioallantoic membrane (CAM). Microsprouting induced by VEGF in the rat aortic ring and blood vessel formation in a mouse Matrigel plug were individually suppressed by HMJ-30. In an in vitro study, HMJ-30 induced the apoptotic death of HUVECs as indicated by DNA fragmentation and promoted reactive oxygen species (ROS) production as determined by flow cytometric assay. In addition, extrinsic caspase signaling (caspase-8 and -3) was activated in the HMJ-30-treated HUVECs and their inhibitors were applied to assess the signal transduction. We investigated the upstream of the death receptor pathway and further observed that the levels of death receptor 5 (DR5) and phosphorylated c-Jun N-terminal kinase (JNK) signals were upregulated in HUVECs following HMJ-30 challenge, which was confirmed by a JNK-specific inhibitor (SP600125). Hence, HMJ-30-induced endothelial cell apoptosis involved the ROS/JNK-regulated DR5 pathway. In summary, HMJ-30 may provide a potential therapeutic effect for the anti-vascular targeting of angiogenesis during cancer treatment.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to cause apoptosis in several types of malignant tumor cells through its interaction with the death domain-containing receptor, death receptor 5 (DR5). In the present study, we showed that co-treatment with troglitazone (TGZ), a synthetic ligand of peroxisome proliferator-activated receptor γ (PPARγ), and TRAIL synergistically induced apoptosis through DR5 upregulation in human colon cancer DLD-1 cells. TGZ elevated DR5 expression at the promoter level through the CCAAT/enhancer-binding protein homologous protein (CHOP) binding site. These results suggest that combined treatment with TGZ and TRAIL may be promising as a new therapy against malignant tumors.

Numerous human chemoprevention studies have demonstrated that non-steroidal anti-inflammatory drugs (NSAIDs) possess chemopreventive effects against a variety of malignant tumors. However, there have been many clinical studies on aspirin, but not ibuprofen, even though ibuprofen is one of the most clinically and safely used NSAIDs showing potent anti-inflammatory effects. Moreover, we reported that many chemopreventive agents enhance the apoptosis-inducing effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is known to be crucial for cancer prevention. We, therefore, investigated whether ibuprofen enhances the cytocidal effect of TRAIL and found that ibuprofen markedly stimulated the apoptosis-inducing efficacy of TRAIL against human colon cancer HCT116 cells. As detected by western blot analysis and real-time RT-PCR, ibuprofen upregulated the expression of death receptor 5 (DR5), a TRAIL receptor. TRAIL-induced apoptosis enhanced by ibuprofen was effectively decreased by a caspase inhibitor and dominant-negative DR5. Noteworthy, co-treatment of ibuprofen with TRAIL did not enhance apoptosis in normal peripheral blood mononuclear cells (PBMCs). These results demonstrated that ibuprofen and TRAIL synergistically induced apoptosis in human colon cancer HCT116 cells but not in normal PBMCs, raising the possibility that ibuprofen may be promising as a safe chemopreventive agent against colon cancer.

Tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL or Apo2L) is a member of the tumor necrosis factor superfamily that induces apoptosis in various cancer cell types but not in most normal cells. However, it is clear that not all cancer cells are sensitive to the killing effects of TRAIL, including breast cancer. Previous studies have demonstrated that chemotherapeutic drugs sensitize tumor cells to apoptosis induced by TRAIL in several types of malignancies. In the present study, we studied the effects of TRAIL combined with cisplatin on two breast cancer cell lines MDA-MB-468 and HCC-1937 in vitro. MTT assay, crystal violet staining assay, DAPI staining assay and flow cytometric analysis were undertaken to evaluate the enhancement of breast cancer cell death using Ad-sTRAIL and/or cisplatin. The levels of apoptotic molecules in signal transduction pathways were analyzed by real-time RT-PCR and western blotting. We found that co-treatment with Ad-sTRAIL and cisplatin exhibited stronger cytotoxicity and induced more significant apoptosis in breast cancer cells compared with Ad-sTRAIL or cisplatin alone. Pretreatment with cisplatin significantly enhanced the expression of DR5. Moreover, the induction of apoptosis by TRAIL plus cisplatin was accompanied by the downregulation of cFLIP and BCL2L1, and simultaneously robust enzymatic activation of caspase-8, culminating in decreased cancer cell survival. The present study revealed that TRAIL conjugated with cisplatin exhibited a markedly increased cytotoxic and apoptosis-inducing effect on breast cancer cells.

Biofunctional marine compounds have recently received substantial attention for their nutraceutical characteristics. In this study, we investigated the apoptosis-inducing effects of sphingoid bases prepared from sea cucumber using human hepatoma HepG2 cells. Apoptotic effects were determined by cell viability assay, DNA fragmentation assay, caspase-3 and caspase-8 activities. The expression levels of apoptosis-inducing death receptor-5 (DR5) and p-AKT were assayed by western blot analysis, and mRNA expression of bax, GADD45 and PPARγ was assayed by quantitative RT-PCR analysis. Sphingoid bases from sea cucumber markedly reduced the cell viability of HepG2 cells. DNA fragmentation indicative of apoptosis was observed in a dose-dependent manner. The expression levels of the apoptosis inducer protein Bax were increased by the sphingoid bases from sea cucumber. GADD45, which plays an important role in apoptosis-inducing pathways, was markedly upregulated by sphingoid bases from sea cucumber. Upregulation of PPARγ mRNA was also observed during apoptosis induced by the sphingoid bases. The expression levels of DR5 and p-AKT proteins were increased and decreased, respectively, as a result of the effects of sphingoid bases from sea cucumber. The results indicate that sphingoid bases from sea cucumber induce apoptosis in HepG2 cells through upregulation of DR5, Bax, GADD45 and PPARγ and downregulation of p-AKT. Our results show for the first time the functional properties of marine sphingoid bases as inducers of apoptosis in HepG2 cells.

Miconazole (MIC), an antifungal agent, diplays anti‑tumorigenic activity in various types of human cancers, including bladder cancer, yet its mechanism of antitumor action is not well understood. In the present study, we demonstrated that, in a cell viability assay, MIC had a cytotoxic effect on human T24, J82 and TSGH-8301 bladder cancer cells in a dose- and time‑dependent manner, but did not exhibit significant toxicity toward human peripheral blood mononuclear cells. Cell cycle analysis revealed that MIC at concentrations of 25 and 50 µM significantly caused G0/G1 arrest in the TSGH-8301 and T24 cells, respectively. DNA fragmentation, mitochondrial membrane potential and western blot analyses showed that MIC inhibited the growth of these cells by both mitochondrial‑mediated and death receptor (DR5)‑mediated apoptosis pathways. Specifically, MIC increased the protein levels of p21 and p27, but decreased the expression of cyclin E1, CDK2 and CDK4. MIC augmented the expression of DR5, cleaved forms of caspase-3 -8 and -9, poly(ADP‑ribose) polymerase and Bax, decreased the expression of Bcl-2 but increased cytosol levels of cytochrome c. Our results suggest that MIC inhibits the growth of bladder cancer cells through induction of G0/G1 arrest and apoptosis via activation of both the extrinsic and intrinsic apoptotic pathways. MIC is a potential chemotherapeutic agent for treating bladder cancer in humans.

Apigenin is one of the plant-originated flavones with anticancer activities. In this study, apigenin was assessed for its in vitro effects on a human colon carcinoma line (HCT‑116 cells) in terms of anti-proliferation, cell cycle progression arrest, apoptosis and intracellular reactive oxygen species (ROS) generation, and then outlined its possible apoptotic mechanism for the cells. Apigenin exerted cytotoxic effect on the cells via inhibiting cell growth in a dose-time-dependent manner and causing morphological changes, arrested cell cycle progression at G0/G1 phase, and decreased mitochondrial membrane potential of the treated cells. Apigenin increased respective ROS generation and Ca2+ release and thereby, caused ER stress in the treated cells. Apigenin shows apoptosis induction towards the cells, resulting in enhanced portion of apoptotic cells. A mechanism involved ROS generation and endoplasmic reticulum stress was outlined for the apigenin-mediated apoptosis via both intrinsic mitochondrial and extrinsic pathways, based on the assayed mRNA and protein expression levels in the cells. With this mechanism, apigenin resulted in the HCT-116 cells with enhanced intracellular ROS generation and Ca2+ release together with damaged mitochondrial membrane, and upregulated protein expression of CHOP, DR5, cleaved BID, Bax, cytochrome c, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9, which triggered apoptosis of the cells.

Understanding the molecular basis of the differential sensitivity of cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis is required to predict therapeutic outcomes and to improve the effectiveness of TRAIL-based therapy. This study aimed to compare the responsiveness of gastric cancer cells to TRAIL treatment and to investigate the molecular basis of the differential TRAIL sensitivity of four gastric cancer cell lines. The TRAIL sensitivity of the four cell lines was ranked in the following order: SNU-16 ≈ SNU-620 > SNU-5 >> SNU-1. The level of Annexin V binding and the activation profile of caspase-3, -8 and -9 corroborated the differential TRAIL susceptibility of the cell lines. To determine the molecular basis of the differential sensitivity to TRAIL, we examined the expression of signaling components involved in TRAIL-mediated apoptosis. The mRNA level and surface expression of death receptor 4 (DR4) were significantly decreased in the SNU-1 cells compared to the other cell lines. Bid cleavage and X-linked inhibitor of apoptosis (XIAP) degradation were significantly increased in the SNU-16 and SNU-620 cells compared to the SNU-5 and SNU-1 cells, although Bid and XIAP were expressed at similar levels across the four cell lines. The expression and degradation of FLICE-inhibitory protein (FLIP) upon TRAIL treatment was independent of TRAIL sensitivity. In conclusion, the differential susceptibility of the four gastric cancer cells to TRAIL may be ascribed to the differential expression of DR4 and the proper augmentation of the death signal by the truncation of Bid and degradation of XIAP.

We investigated the effect of casticin on apoptosis induced by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). We found that casticin potentiated TRAIL-induced apoptosis in human colon cancer cells. Casticin downregulated cell survival proteins including Bcl-xL, Bcl-2, survivin, XIAP and cFLIP, and induced death receptor 5 (DR5), but had no effect on DR4 and decoy receptors (DcR1 or DcR2). Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and casticin. In addition, casticin induced reactive oxygen species (ROS) generation in a dose-dependent manner. Collectively, the present study showed that casticin potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and induction of DR5 mediated by ROS.

TRAIL is a tumor-selective apoptosis-inducing cytokine playing a vital role in the surveillance and elimination of some tumor cells. However, some tumors are resistant to TRAIL treatment. Proteasome inhibitor MG132 exhibits anti-proliferative and pro-apoptotic properties in many tumors. In this study, we demonstrated that proteasome inhibitor MG132 in vitro and in vivo potentiates TRAIL-induced apoptosis in gallbladder carcinoma GBC-SD cells. MG132 was able to inhibit the proliferation of GBC-SD cells and induce apoptosis in a dose-dependent manner. The induction of apoptosis by proteasome inhibitor MG132 was mainly through the extrinsic apoptotic pathways of caspase activation such as caspase-8, caspase-3 and PARP cleavage. In addition, this process was also dependent on the upregulation of death receptor 5 (DR5), which promoted TRAIL-induced apoptosis in GBC-SD cells. Taken together, these findings indicate that MG132 possesses anti-gallbladder cancer potential that correlate with regulation of DR5-dependent pathway, and suggest that MG132 may be a promising agent for sensitizing GBC-SD cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) holds promise for cancer therapy due to its unique capacity to selectively trigger apoptosis in cancer cells. However, TRAIL therapy is greatly hampered by its resistance. A preclinical successful strategy is to identify combination treatments that sensitize resistant cancers to TRAIL. In the present study, we fully assessed TRAIL sensitivity in 9 gastric cancer cell lines. We found combined administration of paclitaxel (PTX) markedly enhanced TRAIL-induced apoptosis in resistant cancer cells both in vitro and in vivo. The sensitization to TRAIL was accompanied by activation of mitochondrial apoptotic pathway, upregulation of TRAIL receptors and downregulation of anti-apoptotic proteins including C-IAP1, C-IAP2, Livin and Mcl-1. Noticeably, we found PTX could suppress the activation of mitogen-activated protein kinases (MAPKs). Inhibition of MAPKs using specific inhibitors (ERK inhibitor U0126, JNK inhibitor SP600125 and P38 inhibitor SB202190) facilitated TRAIL-mediated apoptosis and cytotoxicity. Additionally, SP600125 upregulated TRAL receptors as well as downregulated C-IAP2 and Mcl-1 suggesting the anti-apoptotic role of JNK. Thus, PTX-induced suppression of MAPKs may contribute to restoring TRAIL senstitivity. Collectively, our comprehensive analyses gave new insight into the role of PTX on enhancing TRAIL sensitivity, and provided theoretical references on the development of combination treatment in TRAIL-resistant gastric cancer.

Carcinoma cells that have acquired drug resistance often exhibit cross-resistance to various other cytotoxic stimuli. Here, we investigated the effects of ursodeoxycholic acid (UDCA), a gastrointestinal tumor-suppressor, on a cisplatin‑resistant SNU601 gastric cancer subline (SNU601/R). While other anticancer drugs, including L-OHP, etoposide, and death ligand TRAIL, had minimal effects on the viability of these resistant cells, they were sensitive to UDCA. The UDCA‑induced reduction in the viability of the SNU601/R cells was accomplished through autophagy while the primary means of cell death in the parental SNU601 cells (SNU601/WT) was apoptosis. Previously, we demonstrated that the UDCA-triggered apoptosis of gastric cancer cells was regulated by a cell surface death receptor, TRAIL-R2/DR5, which was upregulated and re-distributed on lipid rafts. The UDCA stimulation of TRAIL-R2/DR5 also occurred in the SNU601/R cells despite the lack of apoptosis. In the present study, we found that CD95/Fas, another cell surface death receptor, was also translocated into lipid rafts in response to UDCA although it was not involved in the decrease in cell viability. Specifically, raft relocalization of CD95/Fas was triggered by UDCA in the SNU601/WT cells in which apoptosis occurred, but not in the SNU601/R cells where autophagic death occurred. Notably, UDCA reduced ATG5 levels, an essential component of autophagy, in the SNU601/WT, but not in the SNU601/R cell line. Moreover, in CD95/Fas-silenced SNU601/WT cells, UDCA did not decrease ATG5 levels and induced autophagic cell death rather than apoptosis. These results imply that raft‑distributed CD95/Fas may support UDCA-induced apoptosis via downregulation of ATG5 levels, preventing the autophagic pathway. Taken together, these results suggest that UDCA induces both apoptotic and autophagic cell death depending on the intracellular signaling environment, thereby conferring the advantage to overcome drug resistance through apoptotic defects.

The aim of the present study was to examine the relationship between DR4 methylation status and gene expression and to determine whether DR4 methylation status affects TRAIL-induced apoptosis in lung squamous carcinoma cells. MSP, RT-PCR and western blot analysis were applied to detect the methylation status and gene expression. An MTT assay was used to detect the cell proliferation inhibition rate and flow cytometry was utilized to detect the apoptotic rate. The results showed that there was no association of the apoptotic rate with the clinicopathological characteristics for 80.6% of 36 lung squamous carcinoma patients in the methylation status (P>0.05). In the lung squamous carcinoma patients, the probability of DR4 low expression was approximately 58.3%, which may be associated with DR4 promoter methylation. The results also showed that a low expression of DR4 was correlated with the prognosis of patients. The in vitro experiments suggested DR4 genes of H226 and SK-MES-1 cells were in the methylation status and their mRNA and proteins had a low expression. Following intervention with 5-Aza-CdR, the DR4 genes of H226 and SK-MES-1 cells were in the unmethylation status and their mRNA and protein expression was significantly upregulated compared with the pre-interference ones, with differences being statistically significant (P

Non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested as the potential new class of preventive or therapeutic antitumor agents. The aim of the present study was to evaluate the antitumor activity of the novel NSAID, CG100649. CG100649 is a novel NSAID dual inhibitor for COX-2 and carbonic anhydrase (CA)-I/-II. In the present study, we investigated the alternative mechanism by which CG100649 mediated suppression of the colon cancer growth and development. The anchorage‑dependent and -independent clonogenic assay showed that CG100649 inhibited the clonogenicity of human colon cancer cells. The flow cytometric analysis showed that CG100649 induced the G2/M cell cycle arrest in colon cancer cells. Animal studies showed that CG100649 inhibited the tumor growth in colon cancer xenograft in nude mice. Furthermore, quantitative PCR and FACS analysis demonstrated that CG100649 upregulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors (DR4 and DR5) but decreased the expression of decoy receptors (DcR1 and DcR2) in colon cancer cells. The results showed that CG100649 treatment sensitized TRAIL‑mediated growth suppression and apoptotic cell death. The combination treatment resulted in significant repression of the intestinal polyp formation in APCmin/+ mice. Our data clearly demonstrated that CG100649 contains preventive and therapeutic activity for colon cancer. The present study may be useful for identification of the potential benefit of the NSAID CG100649, for the achievement of a better treatment response in colon cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells by engaging the death receptors 4 (DR4) and 5 (DR5). We investigated the effect of chemotherapeutic drugs on DR4-mediated apoptosis in human bladder cancer cells, using a human monoclonal agonistic antibody specific for DR4, mapatumumab. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. Treatment of human bladder cancer T24 cells with mapatumumab in combination with mitomycin C, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with mapatumumab in combination with epirubicin (EPI) had a synergistic cytotoxic effect. Synergy was also obtained in KU7 and RT112 human bladder cancer cells. A synergistic effect was also observed with mapatumumab in combination with pirarubicin. The synergy obtained in cytotoxicity with mapatumumab and EPI was also achieved in apoptosis. EPI markedly increased DR4 expression in the bladder cancer cells at both the mRNA and protein levels. Furthermore, the combination-induced cytotoxicity was significantly suppressed by the DR4:Fc chimeric protein. The combination of EPI and mapatumumab significantly activated the caspase cascade, including caspase-8, -9 and -3, which are the downstream molecules of death receptors. These findings indicate that EPI sensitizes bladder cancer cells to DR4-mediated apoptosis through induction of DR4 and activation of caspases, suggesting that the combination therapy of EPI and mapatumumab may be effective for bladder cancer therapy.

We previously reported that purified vitexin compound 1 (VB1, a neolignan from the seed of Chinese herb Vitex negundo) exhibited antitumor activity in cancer cell lines and xenograft models. In the present study, we examined the molecular mechanisms by which activation of the FOXO3a transcription factor mediated VB1-induced apoptosis in hepatocellular carcinoma (HCC) cells. The effects of VB1 on the proliferation of HCC cell lines HepG2, Hep3B, Huh-7 and human embryo liver L-02 cells were investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic death in HepG2 cells was examined using an enzyme-linked immunosorbent assay (ELISA) detection kit, flow cytometry after propidium iodide (PI) staining, and by DNA agarose gel electrophoresis. Caspase activity was measured using ELISA. The AKT/FOXO3a and ERK/FOXO3a pathways were analyzed using western blotting. VB1 inhibited human HCC cell proliferation in a concentration-dependent manner and increased the percentage of sub-G1 population HepG2 cells. Histone/DNA fragmentation and active caspase-3, -8 and -9 levels increased in a concentration-dependent manner and a DNA ladder was formed. The phosphorylation of AKT and ERK1/2 were inhibited and FOXO3a transcription factor was activated, resulting in apoptotic death. Knockdown of AKT1 by small interfering RNA (siRNA) and the MEK1/2 inhibitor, PD98059, enhanced VB1-induced apoptosis and FOXO3a transcriptional activity. Suppression of FOXO3a expression by siRNA inhibited VB1-induced apoptosis. VB1 induced expression of Bim, TRAIL, DR4 and DR5. Activation of the FOXO3a transcription factor appears to mediate pro-apoptotic effects of VB1 by inhibiting the AKT and ERK pathways.

Tumor hypoxia commonly occurs in solid tumors, and correlates with metastasis. Current cancer therapies are inefficient in curing metastatic disease. Herein, we examined effect of Thai propolis extract and its major constituent, chrysin, on hypoxic survival of 4T1 mouse breast cancer cells in vitro, and investigated its underlying mechanism. In vivo effect of chrysin on metastatic progression of cancer cells was studied, both as a single agent and in combination with another antimetastatic agent, agonistic monoclonal antibody targeting the DR5 TRAIL receptor (DR5 mAb). Thai propolis extract and chrysin decreased survival of 4T1 cells after exposure to hypoxia (1% O2), for 2 days. Immunoblot analysis revealed that chrysin inhibited hypoxia-induced STAT3 phosphorylation without affecting HIF-1α protein level. Chrysin also abrogated hypoxia-induced VEGF gene expression as determined by qRT-PCR. The in vivo effect of chrysin was determined in a spontaneous metastasis mouse model of breast cancer, either alone or in combination with DR5 mAb. Daily oral administration of chrysin in Balb/c mice implanted with 4T1 cells significantly suppressed growth of lung metastatic colonies. Moreover, antimetastatic activity of DR5 mAb was enhanced when given in combination with chrysin. We demonstrate that chrysin has potential in controlling metastatic progression.

The present study evaluated the efficacy of drozitumab, a human monoclonal agonistic antibody directed against death receptor 5 (DR5), as a new therapeutic avenue for the targeted treatment of bone and soft-tissue sarcomas. The antitumour activity of drozitumab as a monotherapy or in combination with Nutlin-3a was evaluated in a panel of sarcoma cell lines in vitro and human sarcoma patient samples ex vivo. Knockdown experiments were used to investigate the central role of p53 as a regulator of drozitumab cytotoxicity. Pre-activation of the p53 pathway through Nutlin-3a upregulated DR5, subsequently sensitising sarcoma cell lines and human sarcoma specimens to the pro-apoptotic effects of drozitumab. Silencing of p53 strongly decreased DR5 mRNA expression resulting in abrogation of drozitumab-induced apoptosis. Our study provides the first pre-clinical evaluation of combination therapy using p53-activating agents with drozitumab to further sensitise sarcomas to the cytotoxic effects of DR5 antibody therapy. Refereed/Peer-reviewed

Neuroendocrine tumors respond poorly to radiation and conventional chemotherapy, hence surgical removal of the neoplastic tissue is still the most effective way of treatment. In an attempt to find new therapeutic plant extracts of Christia vespertilionis (CV) their antitumor potential in human medullary thyroid carcinoma (MTC) and human small intestinal neuroendocrine tumor (SI-NET) cell lines were tested. Proliferation and viability were analyzed using cell counting and WST-1 assay. Apoptosis was determined by microscopy, luminescence assays for caspases 3/7, and expression studies of apoptosis-related genes. CV extracts showed antiproliferative and proapoptotic effects in all MTC and SI-NET cell lines, whereby high growth inhibition was observed by treatment with the ethylacetate-extracts (CV-45) in tumor cell lines but not in normal human fibroblasts. Furthermore CV-45 treatment resulted in alterations of gene expression of PDCD5, MTDH and TNFRSF10b in MTC as well as in SI-NET cells. The results indicate that Christia vespertilionis could serve as an anticancer therapeutic for treatment of neuroendocrine tumors.

Evidence suggests that mistletoe extract has the potential to be used as an anticancer agent. Abnobaviscum F® is a European mistletoe extract from the host tree Fraxinus. We investigated the effect of Abnobaviscum F on the growth and survival of different leukemia cell lines. Abnobaviscum F treatment strongly reduced survival and induced apoptosis of K562 (human myeloid leukemia), RPMI-8226 (human plasmacytoma) and L1210 (murine lymphocytic leukemia) cells in culture. Using K562 cells to further investigate the mechanism of action of Abnobaviscum F, we showed that Abnobaviscum F-induced cell death was associated with the activation of caspase-9, JNK-1/2 and p38 MAPK, as well as with the downregulation of Mcl-1, and inhibition of ERK-1/2 and PKB phosphorylation. Moreover, Abnobaviscum F treatment led to both a reduction of cellular glutathione (GSH) and the induction of ER stress (GRP78 and CHOP induction and eIF-2α phosphorylation). By contrast, Abnobaviscum F did not impact the expression of the DR4 and DR5 death receptors. The Abnobaviscum F-induced apoptosis of K562 cells was blocked by pretreatment with either GSH, z-VAD-fmk or SP600125. Our results, therefore, show that Abnobaviscum F induces apoptosis of K562 cells through the activation of the intrinsic caspase pathway, the phosphorylation of JNK-1, the reduction of cellular GSH, and the induction of ER stress.

Ursodeoxycholic acid (UDCA) has been regarded as a suppressor of gastrointestinal cancer, but the mechanisms underlying its antitumor effects are not fully understood. Previously, we reported the antitumor effect of UDCA by demonstrating that UDCA induces apoptosis of gastric cancer cells. Bile acids are known to activate the ERK pathway and ERK is a representative oncogenic kinase in cancer cells. Here, we investigated the role of ERK in UDCA-induced gastric cancer cell apoptosis. We found that UDCA enhanced the phosphorylation of ERK1/2 and MEK1/2. The prevention of MEK by the pharmacologic inhibitors PD98059 and U0126, resulted in decreased UDCA-induced apoptosis as shown by the reduction of apoptotic body formation, caspase-8 activity, and caspase-3, -6 and PARP cleavage, indicating that ERK exerts pro-apoptotic activity upon exposure to UDCA. In addition, U0126 reduced UDCA-triggered TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5) expression. In gene silencing studies, we observed that RNA interference of ERK2 decreased apoptosis and reduced DR5 overexpression. Lipid raft disrupting agent, methyl-β-cyclodextrin, blunted the phosphorylation of ERK1/2, indicating that ERK activation is regulated in a lipid raft-dependent manner. On the other hand, tumor-promoting bile acid, deoxycholic acid (DCA), also phosphorylated ERK in SNU601 cells. However, the DCA-triggered ERK pathway exerted anti-apoptotic function in the cells. Suppression of the ERK pathway enhanced DCA-induced apoptosis, and ERK activation was observed to be lipid raft-independently controlled. These results indicated that UDCA and DCA may cause differential responses in gastric cancer cells through the ERK signaling molecule. Thus, ERK activation may be a possible mechanism by which UDCA and DCA represent differential activities in gastrointestinal cancer.

Sulindac analogs represent one of the most efficacious groups of NSAIDs reducing the risk of colon cancer. Recent studies have shown that sulindac sulfide, a sulindac analog effective at lower doses compared to its parent compound, triggers the death receptor (DR)5-dependent extrinsic apoptotic pathway. Induction of apoptosis via activation of the DR-mediated pathway would be an ideal therapeutic strategy to eliminate cancer cells. In this study, we investigated the possibility that colon cancer cells are sensitized to sulindac sulfide-induced apoptosis by docosahexaenoic acid (DHA), via activation of the DR/extrinsic apoptotic pathway. Our data demonstrated that DHA combination sensitized colon cancer cells to sulindac sulfide-induced apoptosis, leading to enhanced growth suppression of human colon cancer xenografts. The combination effect was primarily attributed to increased cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-8 activation. Moreover, pretreatment with z-IETD-FMK (caspase-8 inhibitor) or stable expression of dominant negative caspase-8 genes blocked DHA/sulindac sulfide cotreatment-induced apoptosis. In view of the finding that DR5 silencing abrogated the combination-stimulated apoptosis, we propose that apoptotic synergy induced by sulindac sulfide plus DHA is mediated via DR5. Our findings collectively support the utility of a combination of sulindac sulfide and DHA in the effective prevention and treatment of colon cancer.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines and has been shown to induce cell apoptosis in many types of tumors, but not in normal cells. This tumor-selective property has made TRAIL a promising approach for the development of cancer therapy. However, hepatocellular carcinoma (HCC) cells display a striking resistance to TRAIL. Although some chemotherapeutic agents can overcome this resistance, safety issues remain a concern because the combination of these agents and TRAIL has been reported to induce toxicity in normal hepatocytes. In this study, we examined whether cisplatin could reverse TRAIL resistance in HCC cells with different p53 status and evaluated the toxicity of combination TRAIL and cisplatin to normal hepatocytes and mesenchymal stem cells (MSCs). We observed that cisplatin could efficiently sensitize HCC cells, but not hepatocytes and MSCs to TRAIL-induced apoptosis within a wide therapeutic window. The apoptosis of HCC cells only partially depended on the upregulation of DR5 and the status of p53. In addition, we provide favorable evidence supporting the feasibility of the combination of chemotherapy and MSCs transduced with TRAIL.

The present data showed that a novel synthesized compound, 6-acetyl-9-(3,4,5-trimethoxybenzyl)-9H-pyrido [2,3-b]indole (HAC-Y6), exhibited potent antitumor activity against human hepatocellular carcinoma (HCC) cells in vitro. Western blot and immunofluorescence experiments showed that HAC-Y6 depolymerized microtubules similarly to the effects of colchicine. HAC-Y6-treatment in Hep3B cells resulted in the accumulation of the G2/M phase and induced apoptosis. In addition, HAC-Y6-treatment influenced the expression of cell cycle and apoptosis related proteins in Hep3B cells. HAC-Y6 exposure increased caspases-3, -8, -9 and Bax protein levels, while reducing levels of Bcl-2 family proteins. Moreover, Bid, a substrate of caspase-8, was also activated by HAC-Y6. Treatment of cells caused the up-regulation of the death receptor 4 (DR4) and phosphorylation of p38. Taken together, we show that HAC-Y6 exhibited its antitumor activity by disrupting microtubule assembly, causing cell cycle arrest and apoptosis through both extrinsic and intrinsic pathways in Hep3B cells. Therefore, the novel compound HAC-Y6 is a promising microtubule inhibitor that has great potential for treatment of HCC.

A component from Emilia sonchifolia (L.) DC, γ-humulene, was investigated. Significantly decreased cell viability of human colorectal cancer HT29 cells in a dose-dependent manner with IC50 53.67±2.99 μM for 24-h treatment was found. γ-Humulene induced apoptotic cell death and apoptosis was confirmed by morphological assessment. The staining with propidium iodide (PI) and flow cytometric analysis also showed that γ-humulene significantly promoted the sub-G1 phase (an apoptotic population) in HT29 cells. Colorimetric assays indicated that pretreatment with a specific inhibitor of caspase-8 (Z-IETD-FMK) significantly reduced activities of caspase-8 and caspase-3 in examined HT29 cells. γ-Humulene stimulated the death receptor 5 (DR5), DR4, Fas-associated protein with death domain (FADD), the cleavage of caspase-8 and cleavage caspase-3, but reduced the protein levels of cellular Fas-associated death-domain-like IL-1s-converting enzyme inhibitory protein (c-FLIP) by Western blot analysis. Consequently, γ-humulene-triggered cell death was significantly attenuated by DR5 siRNA and the caspase-8 inhibitor. Based on our results, we suggest that γ-humulene induced apoptotic cell death in HT29 cells through a DR5-mediated caspase-8 and -3-dependent signaling pathway. Therefore, this agent might be useful for developing new therapeutic regimens for treatment of colorectal cancer in the future.

The detection of molecular targeted agents is a milestone in cancer treatment. Despite the achievements, the efficacy of single targeted agents in combination with radiotherapy is limited by putative treatment resistance. We therefore tested a rationally designed triple therapy consisting of an agonistic antibody against either TRAIL-R1 (mapatumumab/HGS-ETR1) or TRAIL-R2 (lexatumumab/HGS-ETR2) in combination with the established chemotherapeutic drug cisplatin in a panel of solid tumour cell lines derived from head and neck as well as colorectal carcinomas. Induction of apoptosis after monotherapy, double or triple treatment was determined in FaDu (squamous cancer cell line of the head and neck), Colo205 and HCT116 cells (colorectal adenocarcinoma cell lines) by Hoechst 33342 stain. Double and triple therapies were compared using analysis of variance followed by post hoc tests. The degree of interaction was determined by 3D-isobologram analysis. A knockout variant of HCT116 was used to examine Bax-dependence of the triple therapy to gain insight into the underlying molecular signaling pathways possibly responsible for the observed effects. Dose-response relationships revealed different baseline activities of the modalities dependent on cell type. Triple therapy was more effective than double therapy in most cases according to the induction of apoptosis. Furthermore, a synergistic efficacy of the triple therapy was demonstrated in a subset of tumour cell lines. The efficacy of this multimodal approach was highly dependent on the presence of Bax. Our data suggest that targeted agents can be effectively added to existing multimodal therapy approaches which might open new perspectives in radiation oncology.

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in many cancer cells. However, a significant proportion of tumours are TRAIL-resistant erecting a major hurdle for a successful TRAIL-based treatment regimen in the future. In this context, it would be a major advantage to be able to identify the tumours that respond to TRAIL. The existence of two apoptosis-inducing receptors (TRAIL-R1 and TRAIL-R2) and two receptors that cannot transmit an apoptotic signal and have an inhibitory function (TRAIL-R3 and TRAIL-R4) make TRAIL signalling complicated. We analysed the surface expression of all four membrane-bound TRAIL receptors in cancer cell lines of various origin and primary cancer and normal cells and found a good correlation between TRAIL-sensitivity and the expression of TRAIL-R1 alone, but an even better correlation when a ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4 was analysed. Experimental overexpression of TRAIL-R1 alone or in combination with TRAIL-R4 in PANC-1 cells confirmed our correlation results. Similar to the surface expression-apoptosis correlation analysis we found a high correlation between TRAIL-sensitivity and the mRNA level ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4. A value of

This study investigated the expression of TRAIL and its receptors in human gastric cancer and normal gastric tissues, the effects of rh-TRAIL with or without chemotherapeutic drugs in apoptosis of the gastric cancer cell line SGC7901 and the expression changes of DR4 and DR5 in SGC7901 cells influenced by chemotherapeutic drugs. The expression of TRAIL, DR4 and DcR1 were studied in 34 cases of human gastric cancer tissues and 15 cases of adjacent normal mucosa tissues, as well as 21 cases of distant normal mucosa tissues by means of immunohistochemistry. In addition, the expression of FasL were studied in gastric cancer tissues by immunohistochemistry. The effects of treatment with rh-TRAIL alone and/or chemotherapeutic drugs on SGC7901 cell growth inhibition were measured by MTT assay and the mRNA changes of DR4 and DR5 were detected by RT-PCR technique. The expression of TRAIL, DR4 and DcR1 in gastric cancer were lower than those of normal tissues (P

TRAIL induces apoptosis in a variety of tumorigenic and transformed cell lines, but not in many normal cells. Recent studies have demonstrated that death receptor 5 (DR5), one of the two death receptors bound by TRAIL, showed expression in most malignantly transformed cells. This study evaluated effects of a monoclonal antibody (TRA-8) to human death receptor 5, combined with ionizing radiation, on radioresistant human larynx squamous carcinoma cell line (Hep-2R). Cells were treated with TRA-8 alone or in combination with radiation, cell viability inhibition was measured by MTT assay, and the induction of apoptosis was determined by Annexin V staining. Radionsensitivity of Hep-2R cells treated with TRA-8 were investigated with long-term clonogenic assays. Regulation of DR5 expression in cells after radiation was analyzed by indirect immunofluorescence using murine TRA-8 in combination with flow cytometry. The results suggested that TRA-8 enhanced radionsensitivity of Hep-2R cells, and that TRA-8 regulated Hep-2R cell cycle arrest at G2/M phase. Irradiation up-regulated the expression of DR5, and when combined with TRA-8 yielded optimal survival benefit. Therefore, TRA-8 can be used in combination with irradiation in radioresistant human larynx squamous carcinoma cells. Monoclonal antibodies such as TRA-8 may play an important role in the development of an effective treatment strategy for patients with radioresistant cancers.

In light of the poor prognosis for ovarian cancer, research continues for innovative and efficacious treatment modalities. It is now widely accepted that new approaches for the treatment of ovarian cancers are pivotal in further improving prognosis of this disease. Secretory leukoprotease inhibitor (SLPI) is an 11.7-kDa non-glycosylated, serine protease inhibitor that has a broad inhibitory spectrum against serine protease. SLPI showed potential therapeutic inhibitory effects mediated by tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), TNF-alpha, death receptor (DR)-4, DR-5 and TNF receptor (TNFR)-I expression which lead to an activation of apoptosis pathway through Caspase-2, Caspase-8 and Caspase-9. We examined whether levels of SLPI protein expression correlated with clinicopathological characteristics in 58 ovarian cancer samples, and investigated the role of SLPI and its biological functions. SLPI expression showed a significant correlation between low expression of SLPI and amount of ascites (p=0.021), lymph node metastasis (p=0.011). We propose that SLPI could be considered a therapeutic target for the treatment of ovarian cancer.

The tumor suppressor protein p53 restricts proliferation in response to DNA damage or the deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival and in some settings promote genomic instability and resistance to certain anti-cancer drugs. It is very important to identify chemotherapeutic agents that activate in a p53-independent manner for the development of treatments for p53-deficient tumors. Pectenotoxin-2 (PTX-2), isolated from marine sponges has been reported to display significant cytotoxicity to p53-deficient cancer cell lines. In this study, we compared the anti-cancer activity of PTX-2 in order to further test the status of p53 using two well-known hepatocarcinoma cell lines, p53-deficient Hep3B and p53-wild-type HepG2. MTT assay indicated that Hep3B cells were highly susceptible, whereas HepG2 cells were more resistant to this compound which was connected with the induction of apoptotic cell death in p53-deficient Hep3B cells, though not in HepG2 cells. The apoptosis induced by PTX-2 in Hep3B cells was associated with the down-regulation of anti-apoptotic Bcl-2 members (Bcl-2 and Bcl-xL) and IAP family proteins, the up-regulation of pro-apoptotic Bax protein and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-receptor 1/receptor 2 (DR4/DR5) and mitochondrial dysfunction. PTX-2 activated caspases (caspase-3, -8 and -9) and the blockade of caspase-3 activity by the caspase-3 inhibitor prevented the PTX-2-induced apoptosis in Hep3B cells. Additionally, the transcription factor early growth response-1 (Egr-1) gene was transcriptionally activated and the levels of non-steroidal anti-inflammatory drugs (NSAID)-activated gene-1 (NAG-1) protein were also elevated in PTX-2-treated Hep3B cells. Although further studies are needed to prove that an increased expression of Egr-1 by PTX-2 directly leads to NAG-1 induction and then apoptosis induction in p53-deficient Hep3B cells, the results of this study suggest that PTX-2 may be a good candidate for the development of a potential anti-tumorigenic agent in p53-deficient tumors.

We compared the response of normal (FHC) and cancer (HT-29) human colon epithelial cells to the important apoptotic inducers TNF-alpha, anti-Fas antibody and TNF-related apoptosis inducing ligand (TRAIL). The two cell lines did not respond to TNF-alpha (15 ng/ml), expressed a limited sensitivity to anti-Fas antibody (200 ng/ml) and a different response to TRAIL (100 ng/ml). We studied apoptosis with regard to the changes at the receptor level (DR, DcR and FLIP) and at the level of mitochondria (Bid protein cleavage, Apo2.7 protein expression and caspase-9 activation). Two different approaches were used to sensitize the cells to TRAIL-induced apoptosis: inhibition of protein synthesis (cycloheximide, CHX) and inhibition of the pro-survival MEK/ERK pathway (U0126). While the two cell lines were markedly sensitized to all three TNF family members by CHX, a different degree of response (especially for TRAIL) was obtained when inhibition of the MEK/ERK pathway was achieved. TRAIL-induced apoptosis was significantly enhanced by U0126 co-treatment in the HT-29 cells, but not in the FHC cells. The most significant differences between the HT-29 and FHC cells co-treated with TRAIL and U0126 were demonstrated with regard to the involvement of the mitochondrial apoptotic pathway, suggesting its importance in the regulation of cell sensitivity to the TRAIL-induced apoptosis.

8-Chloro-adenosine (8-Cl-Ado) is an adenosine derivative, which inhibits proliferation and induces apoptosis in various tumor cells. Subtoxic concentration of 8-Cl-Ado sensitizes human hepatoma cells to tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-triggered apoptosis. However, the molecular mechanism by which TRAIL cytotoxicity is amplified by 8-Cl-Ado is unknown. In the present study, we demonstrated by Western blot and real-time PCR that 8-Cl-Ado selectively up-regulated death receptor 5 (DR5), but not death receptor 4 (DR4), at both protein and RNA levels in human hepatoma cell line BEL-7402. Analysis of the transcriptional regulation of DR5 expression by using Dual-Luciferase reporter assay system demonstrated that the 5'-flanking fragment -207 to -145 upstream to the ATG site within the DR5 promoter region was responsible for the 8-Cl-Ado-upregulated DR5 expression. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) confirmed that 8-Cl-Ado treatment facilitated transcription factor Sp1 binding to its cis-element -198/-189 in the DR5 promoter, suggesting that Sp1 is at least one of the 8-Cl-Ado-responsive transcription factors. However, we observed that nuclear factor kappaB (NF-kappaB) activity remained invariable in the cells treated with 8-Cl-Ado. These data allowed us to draw a conclusion that 8-Cl-Ado-enhanced DR5 expression is regulated by Sp1 binding to the -198/-189 cis-element in DR5 promoter without affecting NF-kappaB activity in the hepatoma cells. This study may shed light on further screening the regulators of DR5 expression and developing novel therapeutic drugs for liver cancer.

Death receptor 4 (DR4) is a receptor of the antitumor death ligand, TNF-related apoptosis-inducing ligand (TRAIL), and is considered a promising molecular target for cancer therapy. Here, we show a novel regulation of DR4 protein. Tunicamycin treatment, which is an inducer of endoplasmic reticulum (ER)-stress, generated a lower molecular-weight pattern of DR4, but not DR5 protein in prostate cancer DU145 and PC3 cells. Thus, we termed the small form of DR4 protein, DR4-Small (DR4-S) and the large form, DR4-Large (DR4-L). Using DR4 siRNA, we confirmed that DR4-S also stands for DR4 protein. Other ER-stress inducers, brefeldin A and thapsigargin did not generate DR4-S. On the other hand, these ER-stress inducers increased DR5 protein. Tunicamycin induces ER-stress following the inhibition of N-linked glycosylation. Thus, we examined DR4 protein in cell lysates treated with glycosydase. Glycosydase treatments generated DR4-S protein, similar to tunicamycin. These results indicate that tunicamycin regulates DR4 protein size via inhibition of glycosylation.

IFN-alpha regulates tumor cell growth at least through induction of apoptosis. We have recently demonstrated that IFN-alpha causes apoptosis through upregulation of TNF-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma and U266 myeloma cells. However, other cell lines such as Ramos and RPMI 8226 underwent apoptosis without any apparent involvement of TRAIL following IFN-alpha stimulation. In this study, we examined whether the IFN-alpha-induced upregulation of TRAIL is essential for the induction of apoptosis. IFN-alpha-induced early phase (48 h) of loss of DeltaPsim was substantially prevented in Daudi B lymphoma cells overexpressing the dominant-negative form of Fas-associated death domain (dnFADD) compared with vector control, whereas a late phase (72 h) of DeltaPsim was comparable to the control. The IFN-alpha-induced early phase of apoptosis was also reduced in the dnFADD-expressing cells, while the late phase of apoptosis was unaffected. IFN-alpha-induced upregulation of TRAIL protein in the dnFADD-expressing Daudi or U266 cells was comparable to their control cells, suggesting that FADD is not involved in the IFN-alpha-induced upregulation of TRAIL. Moreover, the early phase of mitochondrial depolarization was severely prevented by the presence of fusion protein of TRAIL receptor 1 and Fc portion of immunoglobulin (TRAIL-R1:Fc) and TRAIL-R2:Fc. Together, IFN-alpha induces apoptosis in a TRAIL-dependent or -independent manner, depending on the course of the apoptotic process.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family and a potent inducer of apoptosis. Gemcitabine, chemotherapeutic agent is well known to be involved in synergistic cytotoxic effect with TRAIL. But the mechanism of this synergistic effect is still unclear. To examine the effects and synergistic mechanism of gemcitabine on TRAIL-induced apoptosis, A549, HCT116 and SNU638 cells were pretreated with gemcitabine and treated with TRAIL protein. Gemcitabine significantly enhanced A549 cell death induced by TRAIL, but it was inhibited by the pan-caspase inhibitor, Z-VAD-fmk. The combined treatment of both induced the activation of caspase-8 and reduced the mitochondrial transmembrane potential (MTP) and also translocated Bax protein and released the cytochrome-c in A549 cells when compared with that of negative control. In addition, the gemcitabine pretreatment up-regulated DR-5 and p53 protein expression in a time-dependent manner, which suggests the possible involvement of the p53 protein as a transcriptional factor for DR-5 up-regulation. Thus, we report our findings that gemcitabine enhanced the TRAIL-induced apoptosis and the apoptotic signals are mediated by DR-5-dependent pathway and mitochondrial pathway. Taken together, gemcitabine enhanced TRAIL-induced apoptosis via DR-5 up-regulation and lowering MTP, and suggest that gemcitabine may be used as a successful chemotherapeutic agent for ligand type tumor therapy combined with TRAIL.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine having potent cytotoxic activity specifically to tumor cells. Agonistic antibodies against TRAIL receptors are currently being explored as anti-cancer therapeutics. Here, we report studies on JKTR-18, a monovalent human monoclonal antibody Fab selected against human recombinant TRAIL receptor 2 (DR5) by phage display technology. It induced cell death in Jurkat and HL60 leukemia cell lines without the need for secondary crosslinkers in vitro. It did not compete with soluble TRAIL (sTRAIL) for binding to DR5, and its combination with sTRAIL resulted in greater cell death than either agent alone. The cell death induced by JKTR-18 included a caspase-independent mechanism. This is the first report of a monovalent antibody fragment against TRAIL receptor that can induce tumor cell death in the absence of a crosslinker.