An Antidepressant Drug Increased TRAIL Receptor-2 Expression and Sensitized Lung Cancer Cells to TRAIL-induced Apoptosis.

BACKGROUND: TRAIL has emerged as a promising therapeutic target due to its ability to selectively induce apoptosis in cancer cells while sparing normal cells. Autophagy, a highly regulated cellular recycling mechanism, is known to play a cell survival role by providing a required environment for the cell. Recent studies suggest that autophagy plays a significant role in increasing TRAIL resistance in certain cancer cells. Thus, regulating autophagy in TRAIL-mediated cancer therapy is crucial for its role in cancer treatment. OBJECTIVE: Our study explored whether the antidepressant drug desipramine could enhance the ability of TRAIL to kill cancer cells by inhibiting autophagy. METHODS: The effect of desipramine on TRAIL sensitivity was examined in various lung cancer cell lines. Cell viability was measured by morphological analysis, trypan blue exclusion, and crystal violet staining. Flow cytometry analysis was carried out to measure apoptosis with annexin V-PI stained cells. Western blotting, rtPCR, and immunocytochemistry were carried out to measure autophagy and death receptor expression. TEM was carried out to detect autophagy inhibition. RESULTS: Desipramine treatment increased the TRAIL sensitivity in all lung cancer cell lines. Mechanistically, desipramine treatment induced death receptor expression to increase TRAIL sensitivity. This effect was confirmed when the genetic blockade of DR5 reduced the effect of desipramine in enhanced TRAIL-mediated cell death. Further investigation revealed that desipramine treatment increased the LC3 and p62 levels, indicating the inhibition of lysosomal degradation of autophagy. Notably, TRAIL, in combination with either desipramine or the autophagy inhibitor chloroquine, exhibited enhanced cytotoxicity compared to TRAIL treatment alone. CONCLUSION: Our findings revealed the potential of desipramine to induce TRAIL-mediated cell death by autophagy impairment. This discovery suggests its therapeutic potential for inducing TRAIL-mediated cell death by increasing the expression of death receptors, which is caused by impairing autophagy.

NT157 inhibits cell proliferation and sensitizes glioma cells to TRAIL-induced apoptosis by up-regulating DR5 expression.

NT157, a small-molecule tyrosine kinase inhibitor, exhibits broad-spectrum anti-tumor activity. However, NT157-mediated inhibition against glioma has not been explored yet. Herein, the anticancer effects and underlying mechanism of NT157 against human giloma growth were evaluated. The results showed that NT157 alone significantly inhibited glioma cells growth in vitro by lunching cell cycle arrest through up-regulating p21 and p27, and down-regulating cell cycle-related factors. NT157 alone also induced significant glioma cells apoptosis, followed by PARP cleavage and caspase-3 activation. Our findings further revealed that NT157 triggered significant DNA damage and dysfunction of PI3K/AKT, MAPKs and EGFR-STAT3 signaling pathways. Addition of several kinases inhibitors effectively abrogated NT157-induced DR5 up-regulation, which further confirmed the significant role of DR5 pathway. Moreover, combined treatment of NT157 and TRAIL showed enhanced apoptosis against U251 and U87 cells. However, Knockdown of DR5 expression significantly attenuated combined treatment-induced PARP cleavage and caspase-3 activation. Importantly, combined administration of NT157 and TRAIL in vivo effectively inhibited glioma xenograft growth of nude mice by inhibiting cell proliferation and angiogenesis, and inducing DNA damage and apoptosis. Taken together, our findings validated the rational design that combined strategy of NT157 and TRAIL to trigger DNA damage and apoptosis by up-regulating DR5 could be a high efficient way to combat human glioma.

Thymoquinone Crosstalks with DR5 to Sensitize TRAIL Resistance and Stimulate ROS-Mediated Cancer Apoptosis.

OBJECTIVE: Cancer treatment using a targeted inducer of apoptosis like tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) faced the obstacle of resistance, thus providing a plus drug like Thymoquinone (TQ) could be of great interest to tackle breast cancer cells. The aim of the present work is to examine the genetic modulation impacts of the TRAIL receptors and apoptotic markers upon the combinatorial remedy of TRAIL plus TQ on human breast cancer cell lines. METHODS: To achieve this rationale, the protein content-based cytotoxicity using SRB assay, as well as the genetic expressions of the TRAIL receptors (DR4 and DR5) and apoptotic markers (Bcl-2, Cas-8, and FADD) using real time qRT-PCR technique were preceded against breast cancer MCF-7 and MDA-MB-231 cancerous cell lines. RESULTS: The current study showed that the combination therapy of TQ+TRAIL significantly inhibited the protein content-based proliferation of MDA-MB-231 cells more than MCF-7 cells. The synergistic effect of them significantly up-regulated the genetic expressions of DR4, DR5, Cas-8, and FADD genes and inhibited the genetic expression of the Bcl-2 gene in the proposed cell lines treated for 24 h. The induction of the apoptotic genes using the combined therapy was stimulated by the elevation of the reactive oxygen species (ROS); nitric oxide (NO) and malondialdehyde (MDA) levels. CONCLUSIONS: The synergistic influence between TQ which induced the DR5 and TRAIL, facilitating the connection between TRAIL and its receptors on the cancerous cell membrane. Hence, the proposed combination therapy induced the ROS-mediated apoptotic stimulus.

Luteolin mediated targeting of protein network and microRNAs in different cancers: Focus on JAK-STAT, NOTCH, mTOR and TRAIL-mediated signaling pathways.

There has always been a keen interest of basic and clinical researchers to search for cancer therapeutics having minimum off-target effects and maximum anticancer activities. In accordance with this approach, there has been an explosion in the field of natural products research in the past few decades because of extra-ordinary list of natural extracts and their biologically and pharmacologically active constituents having significant medicinal properties. Apparently, luteolin-mediated anticancer effects have been investigated in different cancers but there is superfluousness of superficial data. Generalized scientific evidence encompassing apoptosis, DNA damage and anti-inflammatory effects has been reported extensively. However, how luteolin modulates deregulated oncogenic pathways in different cancers has not been comprehensively uncovered. In this review we have attempted to focus on cutting-edge research which has unveiled remarkable abilities of luteolin to modulate deregulated oncogenic pathways in different cancers. We have partitioned the review into various sections to separately discuss advancements in therapeutic targeting of oncogenic protein networks. We have provided detailed mechanistic insights related to JAK-STAT signaling and summarized how luteolin inhibited STAT proteins to inhibit STAT-driven gene network. We have also individually analyzed Wnt/ß-catenin and NOTCH pathway and how luteolin effectively targeted these pathways. Mapping of the signaling landscape has revealed that NOTCH pathway can be targeted therapeutically. NOTCH pathway was noted to be targeted by luteolin. We have also conceptually analyzed how luteolin restored TRAIL-induced apoptosis in resistant cancers. Luteolin induced an increase in pro-apoptotic proteins and efficiently inhibited anti-apoptotic proteins to induce apoptosis. Luteolin mediated regulation of non-coding RNAs is an exciting and emerging facet. Excitingly, there is sequential and systematic accumulation of clues which have started to shed light on intricate regulation of microRNAs by luteolin in different cancers. Collectively, sophisticated information will enable us to develop a refined understanding of the multi-layered regulation of signaling pathways and non-coding RNAs by luteolin in different cancers.

Stress-induced TRAILR2 expression overcomes TRAIL resistance in cancer cell spheroids.

The influence of 3D microenvironments on apoptosis susceptibility remains poorly understood. Here, we studied the susceptibility of cancer cell spheroids, grown to the size of micrometastases, to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Interestingly, pronounced, spatially coordinated response heterogeneities manifest within spheroidal microenvironments: In spheroids grown from genetically identical cells, TRAIL-resistant subpopulations enclose, and protect TRAIL-hypersensitive cells, thereby increasing overall treatment resistance. TRAIL-resistant layers form at the interface of proliferating and quiescent cells and lack both TRAILR1 and TRAILR2 protein expression. In contrast, oxygen, and nutrient deprivation promote high amounts of TRAILR2 expression in TRAIL-hypersensitive cells in inner spheroid layers. COX-II inhibitor celecoxib further enhanced TRAILR2 expression in spheroids, likely resulting from increased ER stress, and thereby re-sensitized TRAIL-resistant cell layers to treatment. Our analyses explain how TRAIL response heterogeneities manifest within well-defined multicellular environments, and how spatial barriers of TRAIL resistance can be minimized and eliminated.

Benzyl Isothiocyanate Induces Apoptosis via Reactive Oxygen Species-Initiated Mitochondrial Dysfunction and DR4 and DR5 Death Receptor Activation in Gastric Adenocarcinoma Cells.

Benzyl isothiocyanate (BITC) is known to inhibit the metastasis of gastric cancer cells but further studies are needed to confirm its chemotherapeutic potential against gastric cancer. In this study, we observed cell shrinkage and morphological changes in one of the gastric adenocarcinoma cell lines, the AGS cells, after BITC treatment. We performed 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyl-2H-tetrazolium bromide (MTT) assay, a cell viability assay, and found that BITC decreased AGS cell viability. Reactive oxygen species (ROS) analyses using 2',7'-dichlorofluorescin diacetate (DCFDA) revealed that BITC-induced cell death involved intracellular ROS production, which resulted in mitochondrial dysfunction. Additionally, cell viability was partially restored when BITC-treated AGS cells were preincubated with glutathione (GSH). Western blotting indicated that BITC regulated the expressions of the mitochondria-mediated apoptosis signaling molecules, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and cytochrome c (Cyt c). In addition, BITC increased death receptor DR5 expression, and activated the cysteine-aspartic proteases (caspases) cascade. Overall, our results showed that BITC triggers apoptosis in AGS cells via the apoptotic pathways involved in ROS-promoted mitochondrial dysfunction and death receptor activation.

Chrysanthemulide A induces apoptosis through DR5 upregulation via JNK-mediated autophagosome accumulation in human osteosarcoma cells.

Osteosarcoma is the most frequent malignant primary bone tumor, and it generally develops a multidrug resistance. Chrysanthemulide A (CA) is a sesquiterpenoid from the herb Chrysanthemum indicum that has demonstrated a great anti-osteosarcoma potential. In this study, CA-induced apoptotic cell death resulted in the activation of the caspase-8-mediated caspase cascade, as evidenced by the cleavage of the substrate protein Bid and the caspase-8 inhibitor Z-VAD-FMK. The CA treatment upregulated the expression of death receptor 5 (DR5) in both whole cells and the cell membrane. Blocking DR5 expression by the small interfering RNA (siRNA) treatment decreased the caspase-8-mediated caspase cascade and efficiently attenuated CA-induced apoptosis, suggesting the critical role of DR5 in CA-induced apoptotic cell death. CA-induced upregulation of the DR5 protein was accompanied by the accumulation of LC3B-II, indicating the formation of autophagosomes. Importantly, DR5 upregulation was mediated by transcriptionally controlled autophagosome accumulation, as blockade of autophagosomes by LC3B or ATG-5 siRNA substantially decreased DR5 upregulation. Furthermore, CA activated the c-Jun N-terminal kinase (JNK) signaling pathway, and treatment with JNK siRNAs or inhibitor SP600125 significantly attenuated CA-mediated autophagosome accumulation and DR5-mediated cell apoptosis. Finally, CA sensitized the osteosarcoma cells to the DR5 ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic cell death. Above all, these results suggest that CA induces apoptosis through upregulating DR5 via JNK-mediated autophagosome accumulation and that combined treatment with CA and TRAIL might be a promising therapy for osteosarcoma.

Andrographolide sensitizes prostate cancer cells to TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for anticancer therapy. The identification of small molecules that can establish the sensitivity of prostate cancer (PCa) cells to TRAIL-induced apoptosis is crucial for the targeted treatment of PCa. PC3, DU145, JAC-1, TsuPr1, and LNCaP cells were treated with Andrographolide (Andro) and TRAIL, and the apoptosis was measured using the Annexin V/PI double staining method. Real time-polymerase chain reaction (PCR) and Western blot analysis were performed to measure the expression levels of target molecules. RNA interference technique was used to down-regulate the expression of the target protein. We established a nude mouse xenograft model of PCa, which was used to measure the caspase-3 activity in the tumor cells using flow cytometry. In this research study, our results demonstrated that Andro preferentially increased the sensitivity of PCa cells to TRAIL-induced apoptosis at subtoxic concentrations, and the regulation mechanism was related to the up-regulation of DR4. In addition, it also increased the p53 expression and led to the generation of reactive oxygen species (ROS) in the cells. Further research revealed that the DR4 inhibition, p53 expression, and ROS generation can significantly reduce the apoptosis induced by the combination of TRAIL and Andro in PCa cells. In conclusion, Andro increases the sensitivity of PCa cells to TRAIL-induced apoptosis through the generation of ROS and up-regulation of p53 and then promotes PCa cell apoptosis associated with the activation of DR4.

BAY61-3606 potentiates the anti-tumor effects of TRAIL against colon cancer through up-regulating DR4 and down-regulating NF-κB.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is well known for its ability to preferentially induce apoptosis in malignant cells without causing damage to most normal cells. However, inherent and acquired resistance of tumor to TRAIL-induced apoptosis limits its therapeutic applicability. Here we show that the orally available tyrosine kinase inhibitor, BAY61-3606, enhances the sensitivity of human colon cancer cells, especially those harboring active mutations in Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) gene, to TRAIL-induced apoptosis in vitro and in vivo. The sensitization was achieved by up-regulating death receptor 4 (DR4) and the tumor suppressor p53. BAY61-3606-induced the up-regulation of DR4 is p53-dependent. Knockout of p53 decreased BAY61-3606-induced DR4 expression and inhibited the effect of BAY61-3606 on TRAIL-induced apoptosis. In addition, BAY61-3606 suppressed activity of NF-κB and regulated its gene products, which might also contribute to TRAIL-induced apoptosis. In conclusion, our results showed that BAY61-3606 sensitizes colon cancer cells to TRAIL-induced apoptosis via up-regulating DR4 expression in p53-dependent manner and inhibiting NF-κB activity, suggesting that the combination of TRAIL and BAY61-3606 may be a promising therapeutic approach in the treatment of colon cancer.

Boesenberols, Pimarane Diterpenes with TRAIL-Resistance-Overcoming Activity from Boesenbergia pandurata.

TRAIL is a potent and selective inducer of apoptosis in most cancer cells while sparing normal cells, which makes it an attractive target for the development of new cancer therapies. In a screening program on natural resources with the ability to abrogate TRAIL resistance, the bioassay-guided fractionation of Boesenbergia pandurata rhizomes resulted in the isolation of 17 pimarane diterpenes and a monoterpene. Among these, compounds 1-8, named boesenberols A-H, are new pimarane diterpenes. All compounds exhibited TRAIL-resistance-overcoming activity in TRAIL-resistant AGS cells. Subtoxic doses of the major compound 9 sensitized AGS cells to TRAIL-induced apoptosis by up-regulating apoptosis-inducing proteins, such as DR4, DR5, p53, Fas, CHOP, Bak, and cleaved caspases-3, -8, and -9, and down-regulating the levels of cell survival proteins, such as Bcl-2, c-FLIP, and GSK-3ß, in TRAIL-resistant AGS cells. Furthermore, compound 9 did not decrease the viability of noncancerous (HEK293) cells at concentrations up to 30 µM.

Activating Transcription Factor 4 (ATF4)-ATF3-C/EBP Homologous Protein (CHOP) Cascade Shows an Essential Role in the ER Stress-Induced Sensitization of Tetrachlorobenzoquinone-Challenged PC12 Cells to ROS-Mediated Apoptosis via Death Receptor 5 (DR5) Signaling.

Tetrachlorobenzoquinone (TCBQ) is a downstream metabolite of pentachlorophenol (PCP). Previously, we demonstrated that TCBQ caused cytotoxicity due to mitochondrial-related apoptosis. Here, we confirmed the upregulation of death receptor 5 (DR5) followed by the construction of the death-inducing signaling complex (DISC). We also detected the activation of the caspase cascade, which was correlated with TCBQ-induced apoptotic cell death in PC12 cells. The upregulation of DR5 included transcriptional activation and de novo protein synthesis in response to TCBQ. We also identified the endoplasmic reticulum (ER) as a new target for the TCBQ challenge in PC12 cells. The protein kinase R-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α)-mediated activating transcription factor 4 (ATF4)-ATF3-C/EBP homologous protein (CHOP) signaling pathway contributed to the process of TCBQ-induced ER stress. Blocking ATF4, ATF3, or CHOP signaling by gene silencing technology resulted in decreased cell apoptosis after exposure to TCBQ. Finally, NAC ameliorated TCBQ-induced apoptosis and ER stress, which illustrated that TCBQ-induced apoptosis is somehow ROS-dependent. In summary, this study provided important mechanistic insight into how TCBQ utilizes ER stress-related signaling to exhibit pro-apoptotic activity in PC12 cells.

Morusin Induces TRAIL Sensitization by Regulating EGFR and DR5 in Human Glioblastoma Cells.

Glioblastoma is one of the most malignant primary tumors, and the prognosis for glioblastoma patients remains poor. Tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) is considered a promising anticancer agent due to its remarkable ability to selectively kill tumor cells. However, since many cancers are resistant to TRAIL, strategies to overcome resistance are required for the successful use of TRAIL in the clinic. In the present study, the potential of morusin as a TRAIL sensitizer in human glioblastoma cells was evaluated. Treatment with TRAIL or morusin alone showed weak cytotoxicity in human glioblastoma cells. However, combination treatment of TRAIL with morusin synergistically decreased cell viability and increased apoptosis compared with single treatment. Morusin induced expression of death receptor 5 (DR5), but not DR4 or decoy receptors (DcR1 and DcR2). Furthermore, morusin significantly decreased anti-apoptotic molecules survivin and XIAP. In addition, morusin reduced expression of EGFR and PDFGR as well as phosphorylation of STAT3, possibly mediating down-regulation of survivin and XIAP. Together these results suggest that morusin enhances TRAIL sensitivity in human glioblastoma cells through regulating expression of DR5 and EGFR. Therefore, the combination treatment of TRAIL and morusin may be a new therapeutic strategy for malignant glioma patients.

TRAIL and TRAIL receptors splice variants during long-term interferon ß treatment of patients with multiple sclerosis: evaluation as biomarkers for therapeutic response.

OBJECTIVE: We aimed to assess the effects of interferon ß (IFNß) treatment on the expression of the splice variants of the Tumour necrosis factor-Related Apoptosis Inducing Ligand (TRAIL) and its receptors in different cell subpopulations (CD14+, CD4+ and CD8+) from patients with multiple sclerosis (MS), and to determine whether this expression discriminated responders from non-responders to IFNß therapy. METHODS: We examined mRNA expression of the TRAIL and TRAIL receptors variants in patients with MS, at baseline and after one year of IFNß therapy, according to responsiveness to this drug. RESULTS: Long-term therapy with IFNß increased the expression of TRAIL-α in T cell subsets exclusively from responders and decreased the expression of the isoform 2 of TRAILR-2 in monocytes from responders as well as non-responders. Lower expression of TRAIL-α, and higher expression of TRAIL-ß in monocytes and T cells, was found before the onset of IFNß therapy in patients who will subsequently become responders. Baseline expression of TRAILR-1 was also significantly higher in monocytes and CD4+ T cells from responders. CONCLUSIONS: The present study shows that long-term IFNß treatment has a direct influence on TRAIL-α and TRAILR-2 isoform 2 expression. Besides, receiver operating characteristic analysis revealed that the baseline expression of TRAIL-α in monocytes and T cells, and that of TRAILR-1 in monocytes and CD4+ T cells, showed a predictive value of the clinical response to IFNß therapy, pointing to a role of TRAIL system in the mechanism of action of IFNß in MS that will need further investigation.

FTY720 enhances TRAIL-mediated apoptosis by up-regulating DR5 and down-regulating Mcl-1 in cancer cells.

FTY720, Fingolimod, is a functional antagonist to the sphingosine-1-phosphate (S1P) receptor and an inhibitor of sphingosine kinase 1. Here, we showed that a combination of FTY720 and TRAIL induced apoptosis in human renal, breast, and colon carcinoma cells. Most importantly, this combination had no effect on normal cells. Furthermore, the combined treatment with FTY720 and TRAIL reduced tumor growth in xenograft models. FTY720 up-regulated death receptor (DR)5 at post-translational level. Knockdown of DR5 markedly blocked apoptosis induced by the combined treatment. FTY720 also inhibited Mcl-1 expression at the post-translational level. Over-expression of Mcl-1 blocked apoptosis induced by FTY720 and TRAIL. Interestingly, phospho-FTY720 and inhibitors of sphingosine kinase failed to enhance TRAIL-induced apoptosis. Thus, FTY720 enables TRAIL-induced apoptosis through up-regulation of DR5 and down-regulation of Mcl-1 in human cancer cells.

Scopadulciol, Isolated from Scoparia dulcis, Induces ß-Catenin Degradation and Overcomes Tumor Necrosis Factor-Related Apoptosis Ligand Resistance in AGS Human Gastric Adenocarcinoma Cells.

Scopadulciol (1), a scopadulan-type diterpenoid, was isolated from Scoparia dulcis along with three other compounds (2-4) by an activity-guided approach using the TCF reporter (TOP) luciferase-based assay system. A fluorometric microculture cytotoxicity assay (FMCA) revealed that compound 1 was cytotoxic to AGS human gastric adenocarcinoma cells. The treatment of AGS cells with 1 decreased ß-catenin levels and also inhibited its nuclear localization. The pretreatment of AGS cells with a proteasome inhibitor, either MG132 or epoxomicin, protected against the degradation of ß-catenin induced by 1. The 1-induced degradation of ß-catenin was also abrogated in the presence of pifithrin-α, an inhibitor of p53 transcriptional activity. Compound 1 inhibited TOP activity in AGS cells and downregulated the protein levels of cyclin D1, c-myc, and survivin. Compound 1 also sensitized AGS cells to tumor necrosis factor-related apoptosis ligand (TRAIL)-induced apoptosis by increasing the levels of the death receptors, DR4 and DR5, and decreasing the level of the antiapoptotic protein Bcl-2. Collectively, our results demonstrated that 1 induced the p53- and proteasome-dependent degradation of ß-catenin, which resulted in the inhibition of TCF/ß-catenin transcription in AGS cells. Furthermore, 1 enhanced apoptosis in TRAIL-resistant AGS when combined with TRAIL.

Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.

Epigenetic marks responsible for cadmium-induced melanoma cell overgrowth.

Cadmium (Cd) is a human carcinogen that likely acts via epigenetic mechanisms. However, the precise role of Cd in melanoma remains to be defined. The goals of this study are to: (i) examine the effect of Cd on the proliferation rate of cutaneous and uveal melanoma cells; (ii) identify the genes affected by Cd exposure; (iii) understand whether epigenetic changes are involved in the response to Cd. The cell growth capacity increased at 48 h after Cd treatment at doses ranging from 0.5 to 10 µM. The research on the key genes regulating proliferation has shown that aberrant methylation is responsible for silencing of p16(INK4A) and caspase 8 in uveal and cutaneous melanoma cells, respectively. The methylation and expression patterns of p14(ARF), death receptors 4/5, and E-cadherin remained unmodified after Cd treatment in all the cell lines analyzed. Ectopic expression of p16(INK4A) abolished the overgrowth of uveal melanoma cells in response to Cd and the overexpression of caspase 8 drastically increased the apoptotic rate of Cd-treated cutaneous melanoma cells. In conclusion, our data suggest that hypermethylation of p16(INK4A) and caspase 8 represents the most common event linked to Cd-induced stimulation of cell growth and inhibition of cell death pathway in melanoma.

Complex changes in the apoptotic and cell differentiation programs during initiation of the hair follicle response to chemotherapy.

Chemotherapy has severe side effects in normal rapidly proliferating organs, such as hair follicles, and causes massive apoptosis in hair matrix keratinocytes followed by hair loss. To define the molecular signature of hair follicle response to chemotherapy, human scalp hair follicles cultured ex vivo were treated with doxorubicin (DXR), and global microarray analysis was performed 3 hours after treatment. Microarray data revealed changes in expression of 504 genes in DXR-treated hair follicles versus controls. Among these genes, upregulations of several tumor necrosis factor family of apoptotic receptors (FAS, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) receptors 1/2), as well as of a large number of keratin-associated protein genes, were seen after DXR treatment. Hair follicle apoptosis induced by DXR was significantly inhibited by either TRAIL-neutralizing antibody or caspase-8 inhibitor, thus suggesting a previously unreported role for TRAIL receptor signaling in mediating DXR-induced hair loss. These data demonstrate that the early phase of the hair follicle response to DXR includes upregulation of apoptosis-associated markers, as well as substantial reorganization of the terminal differentiation programs in hair follicle keratinocytes. These data provide an important platform for further studies toward the design of effective approaches for the management of chemotherapy-induced hair loss.

Synergistic antitumor activity of recombinant human Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with carboplatin and pemetrexed in malignant pleural mesothelioma.

INTRODUCTION: Malignant pleural mesothelioma (MPM) is an aggressive, currently incurable tumor with increasing incidence in industrialized countries. Tumor necrosis factor-related, apoptosis-inducing ligand (TRAIL) is a member of the TNF family, which induces cancer cell death through extrinsic apoptotic pathway, while sparing normal cells. The aim of this study was to investigate the antitumor activity of recombinant human Apo2L/TRAIL (dulanermin) in combination with chemotherapy in MPM in vitro and in vivo. METHODS: In the present studies, we employed a panel of MPM cell lines to test the antitumor activity of recombinant human Apo2L/TRAIL (T) in combination with carboplatin and pemetrexed (CP) in vitro and SCID mice. RESULTS: Results demonstrated a significant increase of apoptosis in cell lines treated with CPT compared with those receiving CP or T as single agents. This synergistic effect was dependent on the ability of CP to increase the expression of the TRAIL receptors DR4 and DR5 in a p53 manner. The CPT combination was also effective in blocking the growth of MPM cell lines in a SCID mice preclinical model. CONCLUSIONS: CPT increases MPM cell death in vitro and in vivo compared with CP. In vitro results suggest that chemotherapy sensitizes MPM to TRAIL-dependent apoptosis through p53 activation and subsequent upregulation of DRs.

Death receptor 5-targeted depletion of interleukin-23-producing macrophages, Th17, and Th1/17 associated with defective tyrosine phosphatase in mice and patients with rheumatoid arthritis.

OBJECTIVE: Bidirectional interactions between granulocyte-macrophage colony-stimulating factor-positive (GM-CSF+) T cells and interferon regulatory factor 5-positive (IRF-5+) macrophages play a major role in autoimmunity. In the absence of SH2 domain-containing phosphatase 1 (SHP-1), GM-CSF-stimulated cells are resistant to death receptor (DR)-mediated apoptosis. The objective of this study was to determine whether TRA-8, an anti-DR5 agonistic antibody, can eliminate inflammatory macrophages and CD4 T cells in the SHP-1-deficient condition. METHODS: Ubiquitous Cre (Ubc.Cre) human/mouse-chimeric DR5-transgenic mice were crossed with viable SHP-1-defective motheaten (mev/mev) mice. TRA-8 was administered weekly for up to 4 weeks. The clinical scores, histopathologic severity, and macrophage and CD4 T cell phenotypes were evaluated. The role of TRA-8 in depleting inflammatory macrophages and CD4 T cells was also evaluated, using synovial fluid obtained from patients with rheumatoid arthritis (RA). RESULTS: The levels of inflammatory macrophages (interleukin-23-positive [IL-23+] IRF-5+) and CD4 T cells (IL-17+ GM-CSF+) were elevated in mev/mev mice. In DR5-transgenic mev/mev mice, DR5 expression was up-regulated in these 2 cell populations. TRA-8 treatment depleted these cell populations and resulted in a significant reduction in inflammation and in the titers of autoantibodies. In synovial cells from patients with RA, the expression of IRF5 and DR5 was negatively correlated with the expression of PTPN6. TRA-8, but not TRAIL, suppressed RA inflammatory macrophages and Th17 cells under conditions in which the expression of SHP-1 is low. CONCLUSION: In contrast to TRAIL, which lacks the capability to counteract the survival signal in the absence of SHP-1, TRA-8 eliminated both IRF-5+ IL-23+ M1 macrophages and pathogenic GM-CSF+ IL-17+ CD4 T cells in a SHP-1-independent manner. The results of the current study suggest that TRA-8 can deplete inflammatory cell populations that result from a hyperactive GM-CSF/IRF-5 axis.