SAHA/TRAIL combination induces detachment and anoikis of MDA-MB231 and MCF-7 breast cancer cells

SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERα-positive MCF-7 and the ERα-negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase-8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of ΔΨ(m), activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERK1/2, a kinase which is involved in the phosphorylation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer.     

Cytotoxicity, intracellular distribution and uptake of doxorubicin and doxorubicin coupled to cell-penetrating peptides in different cell lines: A comparative study

One of the major obstacles which are opposed to the success of anticancer treatment is the cell resistance that generally develops after administration of commonly used drugs. In this study, we try to overcome the tumour cell resistance of doxorubicin (Dox) by developing a cell-penetrating peptide (CPP)-anticancer drug conjugate in aim to enhance its intracellular delivery and that its therapeutic effects. For this purpose, two cell-penetrating peptides, penetratin (pene) and tat, derived from the HIV-1 TAT protein, were chemically conjugated to Dox. The cytotoxicity, intracellular distribution and uptake were accessed in CHO cells (Chinese Hamster Ovarian carcinoma cells), HUVEC (Human Umbilical Vein Endothelial Cells), differentiated NG108.15 neuronal cell and breast cancer cells MCF7drug-sensitive or MDA-MB 231 drug-resistant cell lines. The conjugates showed different cell killing activity and intracellular distribution pattern by comparison to Dox as assessed respectively by MTT-based colorimetric cellular cytotoxicity assay, confocal fluorescence microscopy and FACS analysis. After treatment with 3 μM with Dox-CPPs for 2 h, pene increase the Dox cytotoxicity by 7.19-fold in CHO cells, by 11.53-fold in HUVEC cells and by 4.87-fold in MDA-MB 231 cells. However, cytotoxicity was decreased in NG108.15 cells and MCF7. Our CPPs-Dox conjugate proves the validity of CPPs for the cytoplasmic delivery of therapeutically useful molecules and also a valuable strategy to overcome drug resistance.     

Myc Tagging along the TRAIL to Death Receptor 5

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL; also known as Apo2L) is an apoptotic cytokine that is being developed as a novel anticancer agent. TRAIL mediates its effect via death receptors 4 (DR4) and DR5 and appears to selectively induce apoptosis in cancer cells. The molecular basis of why normal cells seem to better tolerate this novel cytokine remains unknown. Recently, it has been reported that Myc oncoprotein by upregulating DR5 appears to augment cellular susceptibility to TRAIL and to DR5 agonistic antibodies. Several previous studies have already established that various clinically relevant agents by upregulating DR5 sensitize cells to TRAIL. However, the finding that DR5 is upregulated by an oncoprotein that is overexpressed in several tumor types is noteworthy and may spark future investigations aiming to explore the Myc and DR5 expression status of primary tumors and their ultimate vulnerability to DR5-targeted therapeutics.     

Transformation by oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by up-regulating death receptor 4 and death receptor 5 through a MEK-dependent pathway

RAS oncogenes play a major role in cancer development by activating an array of signaling pathways, most notably mitogen-activated protein kinases, resulting in aberrant proliferation and inhibition of apoptotic signaling cascades, rendering transformed cells resistant to extrinsic death stimuli. However, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is able to kill specific tumor cells through the engagement of its receptors, death receptor 4 (DR4) and death receptor 5 (DR5), and the activation of apoptotic pathways, providing promising targets for anticancer therapies. In this study, we show that TRAIL induces cell death in human colon adenocarcinoma cells in a MEK-dependent manner. We also report a prolonged MEK-dependent activation of ERK1/2 and increased c-FOS expression induced by TRAIL in this system. Our study reveals that transformation of the colon cell line Caco-2 by Ki- and mainly by Ha-ras oncogenes sensitizes these cells to TRAIL-induced apoptosis by causing specific MEK-dependent up-regulation of DR4 and DR5. These observations taken together reveal that RAS-MEK-ERK1/2 signaling pathway can sensitize cells to TRAIL-induced apoptosis by up-regulating DR4 and DR5 and overall imply that TRAIL-based therapeutic strategies using TRAIL agonists could be used in cases of human colon cancers bearing RAS mutations.     

Nimbolide sensitizes human colon cancer cells to TRAIL through reactive oxygen species- and ERK-dependent up-regulation of death receptors, p53, and Bax

TNF-related apoptosis-inducing ligand (TRAIL) shows promise as a cancer treatment, but acquired tumor resistance to TRAIL is a roadblock. Here we investigated whether nimbolide, a limonoid, could sensitize human colon cancer cells to TRAIL. As indicated by assays that measure esterase activity, sub-G(1) fractions, mitochondrial activity, and activation of caspases, nimbolide potentiated the effect of TRAIL. This limonoid also enhanced expression of death receptors (DRs) DR5 and DR4 in cancer cells. Gene silencing of the receptors reduced the effect of limonoid on TRAIL-induced apoptosis. Using pharmacological inhibitors, we found that activation of ERK and p38 MAPK was required for DR up-regulation by nimbolide. Gene silencing of ERK abolished the enhancement of TRAIL-induced apoptosis. Moreover, our studies indicate that the limonoid induced reactive oxygen species production, which was required for ERK activation, up-regulation of DRs, and sensitization to TRAIL; these effects were mimicked by H(2)O(2). In addition, nimbolide down-regulated cell survival proteins, including I-FLICE, cIAP-1, cIAP-2, Bcl-2, Bcl-xL, survivin, and X-linked inhibitor of apoptosis protein, and up-regulated the pro-apoptotic proteins p53 and Bax. Interestingly, p53 and Bax up-regulation by nimbolide was required for sensitization to TRAIL but not for DR up-regulation. Overall, our results indicate that nimbolide can sensitize colon cancer cells to TRAIL-induced apoptosis through three distinct mechanisms: reactive oxygen species- and ERK-mediated up-regulation of DR5 and DR4, down-regulation of cell survival proteins, and up-regulation of p53 and Bax.     

Involvement of protective autophagy in TRAIL resistance of apoptosis-defective tumor cells

Targeting TRAIL receptors with either recombinant TRAIL or agonistic DR4- or DR5-specific antibodies has been considered a promising treatment for cancer, particularly due to the preferential apoptotic susceptibility of tumor cells over normal cells to TRAIL. However, the realization that many tumors are unresponsive to TRAIL treatment has stimulated interest in identifying apoptotic agents that when used in combination with TRAIL can sensitize tumor cells to TRAIL-mediated apoptosis. Our studies suggest that various apoptosis defects that block TRAIL-mediated cell death at different points along the apoptotic signaling pathway shift the signaling cascade from default apoptosis toward cytoprotective autophagy. We also obtained evidence that inhibition of such a TRAIL-mediated autophagic response by specific knockdown of autophagic genes initiates an effective mitochondrial apoptotic response that is caspase-8-dependent. Currently, the molecular mechanisms linking disabled autophagy to mitochondrial apoptosis are not known. Our analysis of the molecular mechanisms involved in the shift from protective autophagy to apoptosis in response to TRAIL sheds new light on the negative regulation of apoptosis by the autophagic process and by some of its individual components.     

Doxorubicin coupled to penetratin promotes apoptosis in CHO cells by a mechanism involving c-Jun NH2-terminal kinase

Doxorubicin (Dox) has demonstrated potent activity in treating malignant lymphomas but its therapeutic efficacy is hampered by induction of cardiotoxicity. This side effect is related to the ability of the drug to generate reactive oxygen species in cells. Previously, we demonstrated that coupling Dox to penetratin (Pen), a cell penetrating peptide, represent a valuable strategy to overcome drug resistance in CHO cells. In the present study, we evaluated the consequences of the conjugation of Dox to Pen in term of apoptosis induction. When tested on CHO cells, Dox-Pen generated a typical apoptotic phenotype but at lower dose that needed for unconjugated Dox. Cell death induction was associated with chromatin condensation, caspase activation, Bax oligomerisation and release of cytochrome c. By using reactive oxygen species and c-jun NH2-terminal kinase (JNK) inhibitors, we prevented Dox- and Dox-Pen-induced CHO cell death. The chimeric soluble DR5 receptor that inhibits TRAIL induced cell death does not prevent Dox or Dox-Pen-induced cytotoxicity. These observations indicate that conjugation of Dox to cell penetrating peptide does not impair the ability of the drug to trigger cell death through activation of the intrinsic pathway involving c-Jun NH2-terminal kinase but could exhibit less toxic side effects and could warrant its use in clinic.     

Testicular germ cell sensitivity to TRAIL-induced apoptosis is dependent upon p53 expression and is synergistically enhanced by DR5 agonistic antibody treatment

The ability of the TRAIL/DR5 signaling pathway to induce apoptosis has generally been limited to tumor cells. Here we report that in primary testis explants, addition of TRAIL (0.5 μg/ml) caused a three-fold increase in germ cell apoptosis. Furthermore, exposure of C57BL/6 mice to the testicular toxicant, mono-(2-ethylhexyl) phthalate (MEHP), caused an increased p53 stability and elevated DR5 mRNA levels coincident with increases in the levels of apoptosis in spermatocytes. To further assess the mechanisms responsible for the sensitivity of germ cells to undergo TRAIL/DR5-mediated apoptosis, we used the germ cell lines GC-1spg and GC-2spd(ts) (a temperature sensitive spermatocyte-like cell line that allows for p53 nuclear localization at 32°C but not 37°C). Addition of TRAIL and the anti-DR5 monoclonal antibody, MD5-1, triggered a robust synergistic increase of apoptosis in p53 permissive GC-2 cells (32°C) but not in GC-1 cells. In addition, DR5 levels on the plasma membrane of permissive cells were considerably enhanced concomitant with p53 expression and after MD5-1 treatment. These data represent the first indication that testicular germ cells, specifically spermatocytes, can undergo TRAIL-mediated apoptosis and the clinically relevant observation that pretreatment with a DR5 monoclonal antibody can greatly sensitize their apoptotic response to TRAIL. This work was supported, in part, by grants from the National Institute of Environmental Health Sciences/NIH (ES09145, JHR), Toxicology Training grant (ES T32 ES007247, CM), NIH Center Grant (P30 ES07784^, JHR) and the Center for Molecular and Cellular Toxicology (CMCT).     

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane

TRAIL, a ligand of the TNFα family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.     

Celastrol, a Triterpene, Enhances TRAIL-induced Apoptosis through the Down-regulation of Cell Survival Proteins and Up-regulation of Death Receptors

Whether celastrol, a triterpene from traditional Chinese medicine, can modulate the anticancer effects of TRAIL, the cytokine that is currently in clinical trial, was investigated. As indicated by assays that measure plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3, celastrol potentiated the TRAIL-induced apoptosis in human breast cancer cells, and converted TRAIL-resistant cells to TRAIL-sensitive cells. When examined for its mechanism, we found that the triterpene down-regulated the expression of cell survival proteins including cFLIP, IAP-1, Bcl-2, Bcl-xL, survivin, and XIAP and up-regulated Bax expression. In addition, we found that celastrol induced the cell surface expression of both the TRAIL receptors DR4 and DR5. This increase in receptors was noted in a wide variety of cancer cells including breast, lung, colorectal, prostate, esophageal, and pancreatic cancer cells, and myeloid and leukemia cells. Gene silencing of the death receptor abolished the effect of celastrol on TRAIL-induced apoptosis. Induction of the death receptor by the triterpenoid was found to be p53-independent but required the induction of CAAT/enhancer-binding protein homologous protein (CHOP), inasmuch as gene silencing of CHOP abolished the induction of DR5 expression by celastrol and associated enhancement of TRAIL-induced apoptosis. We found that celastrol also induced reactive oxygen species (ROS) generation, and ROS sequestration inhibited celastrol-induced expression of CHOP and DR5, and consequent sensitization to TRAIL. Overall, our results demonstrate that celastrol can potentiate the apoptotic effects of TRAIL through down-regulation of cell survival proteins and up-regulation of death receptors via the ROS-mediated up-regulation of CHOP pathway.     

Lupulone, a hop bitter acid, activates different death pathways involving apoptotic TRAIL-receptors, in human colon tumor cells and in their derived metastatic cells

Our study aimed to compare death signalling pathways triggered by lupulone in TRAIL-sensitive human colon cancer cells (SW480) and in their derived TRAIL-resistant metastatic cells (SW620). Lupulone (40 μg/ml) up-regulated expression of TRAIL DR4/DR5 death receptors at the cell surface of both cell lines, even in the absence of exogenous TRAIL ligand. Cell death induced by lupulone was inhibited in SW480 and SW620 cells exposed to blocking anti-DR4/DR5 antibodies. In SW480 cells, lupulone triggered cell death through a cross-talk between TRAIL-DR4/DR5 and the mitochondrial (intrinsic) pathways involving caspase-8 activation and Bid protein cleavage. As a consequence mitochondrial cytochrome c was released into the cytosol and activation of caspases-9 and -3 was observed. In the metastatic SW620 cells, lupulone restored the sensibility of these cells to TRAIL ligand and activated the extrinsic apoptotic pathway via DR4/DR5 death receptors and the involvement of the caspase-8/caspase-3 cascade. The demonstration that lupulone is able to activate TRAIL-death signalling pathways even in TRAIL resistant cancer cells highlights the potential of this natural compound for cancer prevention and therapy.     

TNF-related apoptosis-inducing ligand as a therapeutic agent in autoimmunity and cancer

Recombinant, soluble TNF-related apoptosis-inducing ligand (TRAIL) is currently being developed as a promising natural immune molecule for trial in cancer patients because it selectively induces apoptosis in transformed or stressed cells but not in most normal cells. In cancer patients, phase 1 and 2 clinical trials using agonistic mAbs that engage the human TRAIL receptors DR4 and DR5 have also provided encouraging results. It is now evident that TRAIL suppresses autoimmune disease in various experimental animal models, suggesting that the therapeutic value of recombinant TRAIL and agonistic DR4 and DR5 mAbs might also extend to the suppression of autoimmune disease. This review provides an insight into our current understanding of the role(s) of TRAIL in disease, with a specific focus on cancer and autoimmunity. We also emphasize biological agents and drugs that sensitize tumour cells to TRAIL-mediated apoptosis and discuss the potential molecular basis for their sensitization.     

Combinatorial treatment with anacardic acid followed by TRAIL augments induction of apoptosis in TRAIL resistant cancer cells by the regulation of p53, MAPK and NFκβ pathways

TRAIL, an apoptosis inducing cytokine currently in phase II clinical trial, was investigated for its capability to induce apoptosis in six different human tumor cell lines out of which three cell lines showed resistance to TRAIL induced apoptosis. To investigate whether Anacardic acid (A1) an active component of Anacardium occidentale can sensitize the resistant cell lines to TRAIL induced apoptosis, we treated the resistant cells with suboptimal concentration of A1 and showed that it is a potent enhancer of TRAIL induced apoptosis which up-regulates the expression of both DR4 and DR5 receptors, which has been observed in the cellular, protein and mRNA levels. The death receptors upregulation consequent to A1 treatment was corroborated by the activation of p53 as well as phosphorylation of p38 and JNK MAP kinases and concomitant inactivation of NFκβ and ERK signaling cascades. Also, A1 modulated the expression of key apoptotic players like Bax, Bcl-2 and CAD along with the abatement of tumor angiogenesis in vivo in EAT mouse model. Thus, post A1 treatment the TRAIL resistant cells turned into TRAIL sensitive cells. Hence our results demonstrate that A1 can synergize TRAIL induced apoptosis through the upregulation of death receptors and downregulation of anti-apoptotic proteins in cancer context.     

Gossypol Induces Death Receptor-5 through Activation of the ROS-ERK-CHOP Pathway and Sensitizes Colon Cancer Cells to TRAIL

Development of resistance to TRAIL, an apoptosis-inducing cytokine, is one of the major problems in its development for cancer treatment. Thus, pharmacological agents that are safe and can sensitize the tumor cells to TRAIL are urgently needed. We investigated whether gossypol, a BH3 mimetic that is currently in the clinic, can potentiate TRAIL-induced apoptosis. Intracellular esterase activity, sub-G₁ cell cycle arrest, and caspase-8, -9, and -3 activity assays revealed that gossypol potentiated TRAIL-induced apoptosis in human colon cancer cells. Gossypol also down-regulated cell survival proteins (Bcl-x_L, Bcl-2, survivin, XIAP, and cFLIP) and dramatically up-regulated TRAIL death receptor (DR)-5 expression but had no effect on DR4 and decoy receptors. Gossypol-induced receptor induction was not cell type-specific, as DR5 induction was observed in other cell types. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by TRAIL and gossypol. Gossypol induction of the death receptor required the induction of CHOP, and thus, gene silencing of CHOP abolished gossypol-induced DR5 expression and associated potentiation of apoptosis. ERK1/2 (but not p38 MAPK or JNK) activation was also required for gossypol-induced TRAIL receptor induction; gene silencing of ERK abolished both DR5 induction and potentiation of apoptosis by TRAIL. We also found that reactive oxygen species produced by gossypol treatment was critical for TRAIL receptor induction and apoptosis potentiation. Overall, our results show that gossypol enhances TRAIL-induced apoptosis through the down-regulation of cell survival proteins and the up-regulation of TRAIL death receptors through the ROS-ERK-CHOP-DR5 pathway.     

<Emphasis Type="Italic">N,N</Emphasis>-Dimethyl phytosphingosine sensitizes HL-60/MX2, a multidrug-resistant variant of HL-60 cells,to doxorubicin-induced cytotoxicity through ROS-mediated release of cytochrome <Emphasis Type="Italic">c</Emphasis> and AIF

Doxorubicin (Dox) is widely used to treat a variety of tumors. However, resistance to this drug is common, making successful treatment more difficult. Previously, we introduced a novel phytosphingosine derivative, N,N-dimethyl phytosphingosine (DMPS), as a potent anticancer therapeutic agent in human leukemia cells. This study was performed to investigate whether DMPS can sensitize HL-60/MX2, a multidrug-resistant variant of HL-60, to Dox-induced apoptosis. Low concentrations of DMPS sensitized HL-60/MX2 cells to Dox-induced apoptosis. Combined Dox + DMPS treatment-induced apoptosis was accompanied by the activation of caspase-8 and caspase-3 as well as PARP cleavage. Cytochrome c and AIF release were also observed in Dox + DMPS-treated HL60/MX2 cells. Pretreatment with z-VAD-fmk markedly prevented caspase-3 activation and moderately suppressed apoptosis, suggesting that Dox + DMPS-induced apoptosis is somewhat (not completely) dependent on caspase. Cytochrome c and AIF release were not affected by pretreatment with z-VAD-fmk. The ROS scavenger NAC efficiently suppressed not only ROS generation, but also caspase-3-mediated PARP cleavage, apoptosis, and release of cytochrome c and AIF, indicating a role of ROS in combined Dox + DMPS treatment-induced apoptotic death signaling. Taken together, these observations suggest that DMPS may be used as a therapeutic agent for overcoming drug-resistance in cancer cells by enhancing drug-induced apoptosis.