Tumor Necrosis Factor-related Apoptosis-inducing Ligand Apoptosis Research Articles

Behind the Adaptive and Resistance Mechanisms of Cancer Stem Cells to TRAIL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), also known as Apo-2 ligand (Apo2L), is a member of the TNF cytokine superfamily. TRAIL has been widely studied as a novel strategy for tumor elimination, as cancer cells overexpress TRAIL death receptors, inducing apoptosis and inhibiting blood vessel formation. However, cancer stem cells (CSCs), which are the main culprits responsible for therapy resistance and cancer remission, can easily develop evasion mechanisms for TRAIL apoptosis. By further modifying their properties, they take advantage of this molecule to improve survival and angiogenesis. The molecular mechanisms that CSCs use for TRAIL resistance and angiogenesis development are not well elucidated. Recent research has shown that proteins and transcription factors from the cell cycle, survival, and invasion pathways are involved. This review summarizes the main mechanism of cell adaption by TRAIL to promote response angiogenic or pro-angiogenic intermediates that facilitate TRAIL resistance regulation and cancer progression by CSCs and novel strategies to induce apoptosis.

Glucose deprivation-induced endoplasmic reticulum stress response plays a pivotal role in enhancement of TRAIL cytotoxicity.

Abnormalities of the tumor vasculature result in insufficient blood supply and development of a tumor microenvironment that is characterized by low glucose concentrations, low extracellular pH, and low oxygen tensions. We previously reported that glucose-deprived conditions induce metabolic stress and promote tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity. In this study, we examined whether the metabolic stress-associated endoplasmic reticulum (ER) stress response pathway plays a pivotal role in the enhancement of TRAIL cytotoxicity. We observed no significant cytotoxicity when human colorectal cancer SW48 cells were treated with various doses of TRAIL (2-100 ng/ml) for 4 h or glucose (0-25 mM) for 24 h. However, a combination of TRAIL and low glucose-induced dose-dependent apoptosis through activation of caspases (-8, -9, and -3). Studies with activating transcription factor 4 (ATF4), C/EBP-homologous protein (CHOP), p53 upregulated modulator of apoptosis (PUMA), or death receptor 5 (DR5)-deficient mouse embryonic fibroblasts or HCT116 cells suggest that the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis are involved in the combined treatment-induced apoptosis. Moreover, the combined treatment-induced apoptosis was completely suppressed in BH3 interacting-domain death agonist (Bid)- or Bcl-2-associated X protein (Bax)-deficient HCT116 cells, but not Bak-deficient HCT116 cells. Interestingly, the combined treatment-induced Bax oligomerization was suppressed in PUMA-deficient HCT116 cells. These results suggest that glucose deprivation enhances TRAIL-induced apoptosis by integrating the ATF4-CHOP-PUMA axis and the ATF4-CHOP-DR5 axis,consequently amplifying the Bid-Bax-associated mitochondria-dependent pathway.

Autophagic Vacuole Secretion Triggered by Chidamide Participates in TRAIL Apoptosis Effect in Breast Cancer Cells.

Breast cancer is one of the most prevalent diseases threatening women's health today. Indepth research on breast cancer (BC) pathogenesis and prevention and treatment methods are gradually receiving attention. Chidamide is a novel histone deacetylase inhibitor (HDACi) that depresses the function of histone deacetylase, consequently affecting the growth of BC cells through epigenetic modification. However, preclinical and clinical studies show that chidamide is ineffective in long-term treatment. We demonstrated in previous experiments that TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis in BC cells and is significantly less non-toxic to normal cells than chidamide. Therefore, in this study, we treated BC cells with chidamide and TRAIL to explore a novel option to reduce the clinical toxicity through augmenting the sensitivity for BC cells. Results from the MTT and cell viability assays indicated that the combination of chidamide and TRAIL in MCF-7 and MDA-MB-231 cells induced BC cell death, while maintaining a reduced concentration of chidamide. Autophagy assay and annexin V analysis showed that the autophagosome microtubuleassociated protein1light chain3-II (LC3-II) was abnormally increased and much more early and late phase of apoptotic cells appeared during chidamide and TRAIL induction. Anti-tumor assays in a BC tumor xenograft model displayed that the mixture of chidamide and TRAIL exhibited stronger effects on inhibiting tumor growth. The data from real-time PCR and western blotting showed that the cytotoxic effect correlated with the expressions of related apoptosis and autophagy factors. Our data are the first to demonstrate the synergistic effects of chidamide and TRAIL in BC cells, specifically, the pharmacological effects on cell death induction. These results lay a solid experimental and theoretical basis to solve the clinical resistance of chidamide.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

Heightened JNK Activation and Reduced XIAP Levels Promote TRAIL and Sunitinib-Mediated Apoptosis in Colon Cancer Models.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis that may be a promising agent in cancer therapy due to its selectivity toward tumor cells. However, many cancer cells are resistant to TRAIL due to defects in apoptosis signaling or activation of survival pathways. We hypothesized that a disruption of pro-survival signaling cascades with the multi-tyrosine kinase inhibitor sunitinib would be an effective strategy to enhance TRAIL-mediated apoptosis. Here we demonstrate that sunitinib significantly augments the anticancer activity of TRAIL in models of colon cancer. The therapeutic benefit of the TRAIL/sunitinib combination was associated with increased apoptosis marked by enhanced caspase-3 cleavage and DNA fragmentation. Overexpression of the anti-apoptotic factor B-cell lymphoma 2 (BCL-2) in HCT116 cells reduced TRAIL/sunitinib-mediated apoptosis, further supporting that sunitinib enhances the anticancer activity of TRAIL via augmented apoptosis. Analysis of pro-survival factors identified that the combination of TRAIL and sunitinib significantly downregulated the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through a c-Jun N-terminal kinase (JNK)-mediated mechanism. Short hairpin RNA (shRNA)-mediated knockdown of JNK confirmed its key role in the regulation of sensitivity to this combination as cells with suppressed JNK expression exhibited significantly reduced TRAIL/sunitinib-mediated apoptosis. Importantly, the therapeutic benefit of the TRAIL/sunitinib combination was validated in the HCT116-Luc and HCT15 colon cancer xenograft models, which both demonstrated significant anti-tumor activity in response to combination treatment. Collectively, our data demonstrate that sunitinib enhances TRAIL-mediated apoptosis by heightened JNK activation, diminished XIAP levels, and augmented apoptosis.

Fenofibrate potentiates chemosensitivity to human breast cancer cells by modulating apoptosis via AKT/NF-κB pathway.

BackgroundCumulatively, evidences revealed that fenofibrate used in the therapy of hyperlipidemia and hypercholesterolemia has anti-cancer effect in multiple cancer types. However, its function and underlying mechanism of chemosensitization in breast cancer remain poorly understood.Materials and methodsThe cytotoxicity of fenofibrate and anti-cancer drugs in breast cancer cells was determined by MTT. Apoptosis and mitochondrial membrane potential were measured using flow cytometry. Caspases and PARP cleavage, the Bcl-2 family members’ protein expression, as well as the activation of AKT and NF-κB signaling pathways were evaluated using Western blot assay. Real-time PCR was used to determine the mRNA expression of Bcl-2 family members.ResultsOur data indicated that fenofibrate suppressed SKBR3 and MDA-MB-231 cell growth in a dose-dependent manner, in the same way as paclitaxel, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), ABT-737, and doxorubicin. Subtoxic levels of fenofibrate significantly augmented paclitaxel, TRAIL, ABT-737, and doxorubicin-induced apoptosis in both these two cell lines. Fenofibrate-promoted chemosensitivity is predominantly mediated by caspase-9 and caspase-3 activation and mitochondrial outer membrane permeabilization. Meanwhile, chemosensitivity promoted by fenofibrate also increased the expression of Bax and Bok and decreased the expression of Mcl-1 and Bcl-xl. Mechanistically, fenofibrate effectively reduced the phosphorylation levels of AKT and NF-κB. In addition, imiquimod, an NF-κB activator, could reverse fenofibrate-induced susceptibility to ABT-737-triggered apoptosis.ConclusionThe present study provided the evidence of the underlying mechanisms on chemosensitization of fenofibrate by inducing the apoptosis of breast cancer in an AKT/NF-κB-dependent manner and implicated the potential application of fenofibrate in potentiating chemosensitivity in breast cancer therapy.

Abstract 4377: The role of Src in TRAIL signaling in non-small cell lung cancer cells

Abstract Introduction: Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and accounts for most cancer-related deaths worldwide. Despite progress in the treatment of subgroups of patients with targeted therapies, better treatments are required to improve overall prognosis. Agents that target the tumor-necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) receptors (TRAIL-R1 and -R2) are able to trigger selective apoptosis in tumor cells, however showed discouraging activity in clinical studies. Resistance to TRAIL-induced apoptosis and non-canonical pro-tumorigenic signaling hampers therapeutic efficacy. Previously, we found that TRAIL induces migration and invasion via RIPK1/Src/STAT3 pathway in apoptosis resistant NSCLC cells. Here we aim to analyze in more detail the role of Src in TRAIL non-canonical signaling and apoptosis resistance. Methods: Cytotoxicity to rhTRAIL was assessed by MTT assays. Src was chemically inhibited or genetically ablated by short hairpin(sh)RNA or CRISPR/CAS9. Src phosphorylation was studied by western blotting. Protein interactions were examined by co-immunoprecipitation (IP) and subsequent western blotting. Src interacting proteins were examined by co-IP experiments in conjunction with LC-mass spectrometric (MS) analyses. Results: The function of Src in TRAIL signaling was examined in TRAIL resistant A549 and sensitive H460 NSCLC cells. rhTRAIL treatment revealed distinct Src phosphorylation patterns, indicating that Src is differentially activated by TRAIL. Subsequently, co-IP experiments were performed showing that in A549 cells Src interacts with RIPK1 and Caspase-8 upon TRAIL treatment, but not in H460 cells. Next, we explored the possible role of Src in regulating TRAIL-induced apoptosis by chemical modulation of Src or by gene silencing or genetic knockout of Src. We found no role for Src in regulating sensitivity or resistance to TRAIL-induced apoptosis. To further investigate possible biological consequences of TRAIL-dependent Src activation we performed co-IP coupled with LC-MS analysis. Abundant differences were found in the Src interactome of A549 and H460 cells and in absence and presence of TRAIL. Various proteins known to be involved in tumor signaling were identified to be in complex with Src, including components of the RAF/MEK/ERK, Wnt and SMAD3 signaling pathways. Currently, mechanistic and validation studies are in progress to elucidate the role of these proteins. Conclusions: Src has no role in controlling sensitivity or resistance to TRAIL-induced apoptosis in the examined NSCLC cells. On the contrary, the Src interactome showed the activation of various pro-tumorigenic pathways by TRAIL. We anticipate that a deeper knowledge of TRAIL signaling will lead to novel therapeutic strategies to improve TRAIL-receptor targeted therapy. Supported by a grant from the Dutch Cancer Society (KWF 2011-5211). Citation Format: Margot de Looff, Steven de Jong, Frank A.E. Kruyt. The role of Src in TRAIL signaling in non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4377.

Monocyte chemotactic protein-induced protein-1 enhances DR5 degradation and negatively regulates DR5 activation-induced apoptosis through its deubiquitinase function.

Monocyte chemotactic protein-induced protein-1 (MCPIP1; also called Regnase-1) encoded by the ZC3H12A gene critically regulates inflammatory responses and immune homeostasis primarily by RNase-dependent and -independent mechanisms. However, the relationship of MCPIP1 with apoptosis and cancer and the underlying mechanisms are largely unclear. The current study has demonstrated a previously uncovered connection between MCPIP1 and the negative regulation of death receptor 5 (DR5; also known as TRAIL-R2 or killer/DR5), a cell surface receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which is produced endogenously by various immune cells such as T cells. Our findings have revealed that MCPIP1 decreases both total cellular and cell surface DR5, primarily through modulating DUB-mediated protein autophagic/lysosomal degradation. Suppression of MCPIP1 by gene knockdown induces the formation of death-induced signaling complex (DISC) and enhances TRAIL or DR5 activation-induced apoptosis in cancer cells. Moreover, we demonstrated an inverse correlation between MCPIP1 expression and DR5 expression/cell sensitivity to DR5 activation-induced apoptosis in cancer cells. Our findings warrant future investigation of the roles of negative regulation of DR5 by MCPIP1 in cancer and in T-cell immunity.

Developing TRAIL/TRAIL death receptor-based cancer therapies.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can initiate the apoptosis pathway by binding to its associated death receptors DR4 and DR5. The activation of the TRAIL pathway in inducing tumor-selective apoptosis leads to the development of TRAIL-based cancer therapies, which include recombinant forms of TRAIL, TRAIL receptor agonists, and other therapeutic agents. Importantly, TRAIL, DR4, and DR5 can all be induced by synthetic and natural agents that activate the TRAIL apoptosis pathway in cancer cells. Thus, understanding the regulation of the TRAIL apoptosis pathway can aid in the development of TRAIL-based therapies for the treatment of human cancer.

Simultaneously targeting Bcl-2 and Akt pathways reverses resistance of nasopharyngeal carcinoma to TRAIL synergistically.

Despite progress in treatment techniques, the five-year survival rate of nasopharyngeal carcinoma (NPC) is disappointing. Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively induce apoptosis in most tumor cells while sparing normal cells. Given the antiapoptotic functions of Bcl-2 and Akt, we examined the effects of targeting these pathways alone or simultaneously on TRAIL apoptosis in NPC cell lines. We first tested the cytotoxic effect of TRAIL and the expression of death receptors, Bcl-2, Akt, and p-Akt on four NPC cell lines by MTT and Western blotting, respectively. Small interfering RNAs (siRNAs) targeting Bcl-2 and PI3-K inhibitor (LY294002) were used alone or combined with TRAIL in the cell lines and cytotoxicity was examined by MTT. Apoptosis rates, mitochondrial transmembrane potential, and apoptotic pathway signals were detected by flow cytometric analysis, DiOC6(3) assays, and Western blotting after the various combination treatments on CNE-2, the cell line that was most resistant to TRAIL. Although no direct correlation between the sensitivity to TRAIL and the relative expression levels of Bcl-2 and activated Akt was found in the NPC cell lines examined, siRNA mediated the downregulation of Bcl-2 and LY294002-induced inactivation of Akt, increasing the sensitivity of all examined NPC cell lines to TRAIL. Synergistic enhancement of TRAIL-mediated cytotoxicity was observed in combination treatment of Bcl-2 siRNA and LY294002 compared to cells treated with each treatment alone. The synergistic effects were mediated through increased apoptotic signaling of the mitochondrial pathway, as was evident from the more increased mitochondrial depolarization, activation of caspase-9 and caspase-3, and suppression of XIAP. This study provides proof of principle that TRAIL combined with simultaneously targeting the Bcl-2 and Akt signaling pathways may have potential as a novel future treatment strategy for NPC.

Pro-apoptotic signaling induced by Retinoic acid and dsRNA is under the control of Interferon Regulatory Factor-3 in breast cancer cells.

Breast cancer is one of the most lethal malignancies for women. Retinoic acid (RA) and double-stranded RNA (dsRNA) are considered signaling molecules with potential anticancer activity. RA, co-administered with the dsRNA mimic polyinosinic-polycytidylic acid (poly(I:C)), synergizes to induce a TRAIL (Tumor-Necrosis-Factor Related Apoptosis-Inducing Ligand)- dependent apoptotic program in breast cancer cells. Here, we report that RA/poly(I:C) co-treatment, synergically, induce the activation of Interferon Regulatory Factor-3 (IRF3) in breast cancer cells. IRF3 activation is mediated by a member of the pathogen recognition receptors, Toll-like receptor-3 (TLR3), since its depletion abrogates IRF3 activation by RA/poly(I:C) co-treatment. Besides induction of TRAIL, apoptosis induced by RA/poly(I:C) correlates with the increased expression of pro-apoptotic TRAIL receptors, TRAIL-R1/2, and the inhibition of the antagonistic receptors TRAIL-R3/4. IRF3 plays an important role in RA/poly(I:C)-induced apoptosis since IRF3 depletion suppresses caspase-8 and caspase-3 activation, TRAIL expression upregulation and apoptosis. Interestingly, RA/poly(I:C) combination synergizes to induce a bioactive autocrine/paracrine loop of type-I Interferons (IFNs) which is ultimately responsible for TRAIL and TRAIL-R1/2 expression upregulation, while inhibition of TRAIL-R3/4 expression is type-I IFN-independent. Our results highlight the importance of IRF3 and type-I IFNs signaling for the pro-apoptotic effects induced by RA and synthetic dsRNA in breast cancer cells.

Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression.

Despite robust antitumor activity in diverse preclinical models, TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have not demonstrated efficacy in clinical trials, underscoring the need to identify agents that enhance their activity. We postulated that the metabolic stress induced by the diabetes drug metformin would sensitize breast cancer cells to TRAIL receptor agonists. Human triple (estrogen receptor, progesterone receptor, and HER2)-negative breast cancer (TNBC) cell lines were treated with TRAIL receptor agonists (monoclonal antibodies or TRAIL peptide), metformin, or the combination. The effects on cell survival, caspase activation, and expression of TRAIL receptors and the antiapoptotic protein XIAP were determined. In addition, XIAP was silenced by RNAi in TNBC cells and the effects on sensitivity to TRAIL were determined. The antitumor effects of metformin, TRAIL, or the combination were evaluated in an orthotopic model of metastatic TNBC. Metformin sensitized diverse TNBC cells to TRAIL receptor agonists. Metformin selectively enhanced the sensitivity of transformed breast epithelial cells to TRAIL receptor agonist-induced caspase activation and apoptosis with little effect on untransformed breast epithelial cells. These effects of metformin were accompanied by robust reductions in the protein levels of XIAP, a negative regulator of TRAIL-induced apoptosis. Silencing XIAP in TNBC cells mimicked the TRAIL-sensitizing effects of metformin. Metformin also enhanced the antitumor effects of TRAIL in a metastatic murine TNBC model. Our findings indicate that metformin enhances the activity of TRAIL receptor agonists, thereby supporting the rationale for additional translational studies combining these agents.

Association of EGFR and KRAS mutations with expression of p-AKT, DR5 and DcR1 in non-small cell lung cancer.

The activation of AKT is one of the causes of resistance to epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKIs). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combines with related receptors to trigger apoptosis or protect the cells against TRAIL apoptosis. This research focused on the association of EGFR and KRAS mutations with expression of AKT, p-AKT, DR5 and DcR1 in non-small cell lung cancer. 82 NSCLC patients were included in the study. xTAG liquichip techonolgy (xTAG-LCT) was applied to investigate the genetic mutation of EGFR and KRAS, Quantitative Real-time PCR was used to test the mRNA expression of AKT, DR5 and DcR1 and Western Blot was applied to test the protein expression of AKT, p-AKT, DR5, and DcR1. We found that of 82 patients, 31 cases had EGFR-activating mutations, more common in female, adenocarcinoma, and non-smoker patients; 9 cases had KRAS mutations, frequently found in patients with smoking history. The expression of AKT and p-AKT correlated with staging, tumor differentiation, and lymph node metastasis. The expression of DR5 in phase III and low differentiation tumor was significantly higher than that in phase I+II and high and median differentiation tumor; the expression of DcR1 in phase III and low differentiation tumor was significantly lower than that in phase I+II and high and median differentiation tumor. Compared with EGFR and KRAS wild type, in NSCLC tissue with EGFR and KRAS mutations, the expression of AKT and p-AKT was significantly higher. These results suggest that EGFR and KRAS mutation status was associated with the expression of AKT and p-AKT. AKT, p-AKT, DR5, and DcR1 all took part in the occurrence and development of NSCLC, and may become a reference index to evaluate the prognosis of NSCLC.

Abstract 3901: Polymeric mechanical amplifiers of tumor cell receptor-mediated apoptosis

Abstract Introduction: It has become evident that tumor cells are responsive to mechanical forces in vivo, and prove critical to tumor cell proliferation and death. Recent work has shown that tumor cells exposed to fluid shear forces increase receptor-mediated signaling. We hypothesized that biocompatible, polymeric particles conjugated to the tumor cell surface act as mechanical amplifiers in presence of fluid shear forces to increase mechanotransduction, and can be exploited to increase the efficacy of therapeutic ligands. Methods: Polymeric particles (size:100 nm-1 μm) were conjugated to free amines on tumor cells via NHS cross linker chemistry. Particles bound to tumor cells were assessed using flow cytometry, brightfield, and confocal fluorescence microscopy. A cone-and-plate viscometer was used to apply a fluid shear force (2.0 dyn/cm⁁2) to tumor cell suspensions and to amplify the force exerted by polymeric particles on tumor cells. Tumor cells (COLO 205, PC-3) were treated with 0.1 μg/mL of a TNF-related apoptosis-inducing ligand (TRAIL) to assess amplified mechanotransduction and receptor-mediated apoptosis in the presence of polymeric particles. An annexin-V apoptosis assay was used to characterize the mode of cell death. Caspase colorimetric assays and inhibitors (Z-VAD-FMK) were utilized to assess caspase-dependence in the mechanotransduction response. Tumor cells were injected into C57/BL6 via tail vein to assess amplification of TRAIL apoptosis in vivo. Results and Discussion: NHS crosslinker chemistry was successfully used to conjugate polymeric particles to the tumor cell surface. In the presence of fluid shear forces, it was found that polymeric particles act to mechanical amplify tumor cell mechanotransduction, as evidenced by increased receptor-mediated apoptosis and decreased tumor cell viability in the presence of the therapeutic ligand TRAIL. Additionally, amplification of TRAIL-mediated apoptosis was increased with particles of increasing size, demonstrating that increasing the force exerted on the cell surface with larger particles amplified the therapeutic response. Annexin-V apoptosis assays showed the addition of conjugated polymeric particles to the cell surfaces nearly doubled tumor cell apoptosis in the presence of TRAIL under shear forces, and inhibition assays revealed the response to be caspase-dependent apoptosis. Polymeric particles amplified TRAIL-mediated tumor cell killing in mice, and reduced both circulating tumor cells in blood and overall tumor cell burden in vivo by over 90%. Conclusions: These data demonstrate that polymeric particles, both degradable and non-degradable, act as mechanical amplifiers of tumor mechanotransduction in the presence of shear forces in vitro and in vivo, and are exploited to increase therapeutic efficacy. Clinically, this approach shows that increased mechanical force applied to target tumor cells can increase sensitivity to therapeutic ligands. Citation Format: Michael J. Mitchell, Robert Langer. Polymeric mechanical amplifiers of tumor cell receptor-mediated apoptosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3901.

A novel, soluble compound, C25, sensitizes to TRAIL-induced apoptosis through upregulation of DR5 expression.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential therapeutic agent that induces apoptosis selectively in tumor cells. However, numerous solid tumor types are resistant to TRAIL. Sensitization to TRAIL has been an area of great research interest, but has met significant challenges because of poor bioavailability, half-life, and solubility of sensitizing compounds such as curcumin. Soluble, TRAIL-sensitizing compounds were screened on the basis of similarity to the redox-active substructure of curcumin and sensitization to TRAIL-induced apoptosis. We determined the effect of the lead compound, C25, in combination with TRAIL in human cancer cell lines using MTS proliferation assays, apoptosis assays, and western blotting. Short hairpin RNA knockdown of death receptor 5 (DR5) was performed to determine whether DR5 upregulation was required for TRAIL-mediated apoptosis. In-vivo efficacy was determined using human lung tumor xenograft models. C25 helped overcome TRAIL resistance by upregulating the expression of the TRAIL receptor DR5 and apoptosis in several tumor cell lines. Blockade of DR5 expression abrogated C25 sensitization to TRAIL, demonstrating the requirement for DR5 upregulation for C25-mediated potentiation of TRAIL-mediated apoptosis. The combination of C25 and TRAIL effectively inhibited tumorigenesis in vivo. This study demonstrates the synergistic efficacy of C25 in sensitization to TRAIL-induced apoptosis in multiple tumor cell types, including highly resistant lung and ovarian tumor cell lines. Furthermore, C25 was efficacious against tumor growth in vivo. Thus, C25 may be a potential therapeutic for cancer in combination with TRAIL or DR5 agonist therapy.

Increased growth-inhibitory and cytotoxic activity of arsenic trioxide in head and neck carcinoma cells with functional p53 deficiency and resistance to EGFR blockade.

Background and PurposeMutations in the p53 gene are frequently observed in squamous cell carcinoma of the head and neck region (SCCHN) and have been associated with drug resistance. The potential of arsenic trioxide (ATO) for treatment of p53-deficient tumor cells and those with acquired resistance to cisplatin and cetuximab was determined.Material and MethodsIn a panel of 10 SCCHN cell lines expressing either wildtype p53, mutated p53 or which lacked p53 by deletion the interference of p53 deficiency with the growth-inhibitory and radiosensitizing potential of ATO was determined. The causal relationship between p53 deficiency and ATO sensitivity was evaluated by reconstitution of wildtype p53 in p53-deficient SCCHN cells. Interference of ATO treatment with cell cycle, DNA repair and apoptosis and its efficacy in cells with acquired resistance to cisplatin and cetuximab was evaluated.ResultsFunctional rather than structural defects in the p53 gene predisposed tumor cells to increased sensitivity to ATO. Reconstitution of wt p53 in p53-deficient SCCHN cells rendered them less sensitive to ATO treatment. Combination of ATO with irradiation inhibited clonogenic growth in an additive manner. The inhibitory effect of ATO in p53-deficient tumor cells was mainly associated with DNA damage, G2/M arrest, upregulation of TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) receptors and apoptosis. Increased activity of ATO was observed in cetuximab-resistant SCCHN cells whereas cisplatin resistance was associated with cross-resistance to ATO.ConclusionsAddition of ATO to treatment regimens for p53-deficient SCCHN and tumor recurrence after cetuximab-containing regimens might represent an attractive strategy in SCCHN.

Role of serum TRAIL level and TRAIL apoptosis gene expression in multiple sclerosis and relation to brain atrophy

One of the presumed pathological mechanisms of multiple sclerosis (MS) is the failure of apoptosis of autoreactive T lymphocytes. This study aimed to determine the relationship of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA gene expression ratio and serum TRAIL levels with MS and brain atrophy. This study was conducted on 53 relapsing-remitting Egyptian MS patients and 25 matched healthy volunteers. The expression of TRAIL in peripheral blood lymphocytes was analyzed by reverse transcription polymerase chain reaction, serum levels of soluble TRAIL (sTRAIL) were determined by enzyme-linked immunosorbent assay and brain MRI measured “black holes” and the bicaudate ratio as a measure of brain atrophy in all patients. The serum TRAIL level was lower in MS patients compared to controls but no difference was seen in the TRAIL mRNA gene expression ratio. No significant correlation was detected between the serum TRAIL level and the TRAIL mRNA expression ratio in either group. No statistically significant correlation was found between serum TRAIL levels or the TRAIL mRNA expression ratio with the number of black holes or the bicaudate ratio on MRI. Apoptosis of T lymphocytes is decreased in MS patients, which could be useful when designing treatments. There was no difference in the TRAIL mRNA gene expression ratio between MS patients and controls.

Helicobacter pylori sensitizes TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human gastric epithelial cells through regulation of FLIP

Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcer and gastric cancer. Apoptosis induced by microbial infections is implicated in the pathogenesis of H. pylori infection. Here we show that human gastric epithelial cells sensitized to H. pylori confer susceptibility to TRAIL-mediated apoptosis via modulation of death receptor signaling. Human gastric epithelial cells are intrinsically resistant to TRAIL-mediated apoptosis. The induction of TRAIL sensitivity by H. pylori is dependent on the activation of caspase-8 and its downstream pathway. H. pylori induces caspase-8 activation via enhanced assembly of the TRAIL death-inducing signaling complex (DISC) through downregulation of cellular FLICE-inhibitory protein (FLIP). Overexpression of FLIP abolished the H. pylori-induced TRAIL sensitivity in human gastric epithelial cells. Our study thus demonstrates that H. pylori induces sensitivity to TRAIL apoptosis by regulation of FLIP and assembly of DISC, which initiates caspase activation, resulting in the breakdown of resistance to apoptosis, and provides insight into the pathogenesis of gastric damage in Helicobacter infection. Modulation of host apoptosis signaling by bacterial interaction adds a new dimension to the pathogenesis of Helicobacter.

Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce apoptosis in many cancer cells without causing toxicity in vivo. However, to date, TRAIL-receptor agonists have only shown limited therapeutic benefit in clinical trials. This can, most likely, be attributed to the fact that 50% of all cancer cell lines and most primary human cancers are TRAIL resistant. Consequently, future TRAIL-based therapies will require the addition of sensitizing agents that remove crucial blocks in the TRAIL apoptosis pathway. Here, we identify PIK-75, a small molecule inhibitor of the p110α isoform of phosphoinositide-3 kinase (PI3K), as an exceptionally potent TRAIL apoptosis sensitizer. Surprisingly, PI3K inhibition was not responsible for this activity. A kinome-wide in vitro screen revealed that PIK-75 strongly inhibits a panel of 27 kinases in addition to p110α. Within this panel, we identified cyclin-dependent kinase 9 (CDK9) as responsible for TRAIL resistance of cancer cells. Combination of CDK9 inhibition with TRAIL effectively induced apoptosis even in highly TRAIL-resistant cancer cells. Mechanistically, CDK9 inhibition resulted in downregulation of cellular FLICE-like inhibitory protein (cFlip) and Mcl-1 at both the mRNA and protein levels. Concomitant cFlip and Mcl-1 downregulation was required and sufficient for TRAIL sensitization by CDK9 inhibition. When evaluating cancer selectivity of TRAIL combined with SNS-032, the most selective and clinically used inhibitor of CDK9, we found that a panel of mostly TRAIL-resistant non-small cell lung cancer cell lines was readily killed, even at low concentrations of TRAIL. Primary human hepatocytes did not succumb to the same treatment regime, defining a therapeutic window. Importantly, TRAIL in combination with SNS-032 eradicated established, orthotopic lung cancer xenografts in vivo. Based on the high potency of CDK9 inhibition as a cancer cell-selective TRAIL-sensitizing strategy, we envisage the development of new, highly effective cancer therapies.

Aldose reductase inhibition enhances TRAIL-induced human colon cancer cell apoptosis through AKT/FOXO3a-dependent upregulation of death receptors

One of the major problems associated with the chemotherapy of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) that selectively kills tumor cells is decreased drug resistance. This warranted the development of safe novel pharmacological agents that could sensitize the tumor cells to TRAIL. Herein, we examined the role of aldose reductase (AR) in sensitizing cancer cells to TRAIL and potentiating TRAIL-induced apoptosis of human colon cancer cells. We demonstrate that AR inhibition potentiates TRAIL-induced cytotoxicity in cancer cells by upregulation of both death receptor (DR)-5 and DR4. Knockdown of DR5 and DR4 significantly (>85%) reduced the sensitizing effect of the AR inhibitor fidarestat on TRAIL-induced apoptosis. Further, AR inhibition also downregulates cell survival proteins (Bcl-xL, Bcl-2, survivin, XIAP, and FLIP) and upregulates the expression of proapoptotic proteins such as Bax and alters mitochondrial membrane potential, leading to cytochrome c release, caspases-3 activation, and PARP cleavage. We found that AR inhibition regulates AKT/PI3K-dependent activation of forkhead transcription factor FOXO3a. Knockdown of FOXO3a significantly (>80%) abolished AR inhibition-induced upregulation of DR5 and DR4 and apoptosis in colon cancer cells. Overall, our results show that fidarestat potentiates TRAIL-induced apoptosis through downregulation of cell survival proteins and upregulation of death receptors via activation of the AKT/FOXO3a pathway.