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

Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

PYCR1 regulates TRAIL‑resistance in non‑small cell lung cancer cells by regulating the redistribution of death receptors.

Although recombinant human TNF-related apoptosis-inducing ligand (TRAIL) protein exhibits antitumor activity in a number of lung and liver cancer cells and tumor-bearing animals, TRAIL resistance has substantially restricted its clinical application. Pyrroline-5-carboxylate reductase 1 (PYCR1) is a key enzyme in the regulation of proline synthesis. PYCR1 is highly expressed in various types of malignant tumor, in which it has been implicated in 5-fluorouracil resistance. However, the possible relationship between PYCR1 and TRAIL resistance remains unclear. In the present study, both reverse transcription-quantitative PCR and western blotting were performed. The results indicated that H1299 cells had higher PYCR1 expression levels and were less sensitive to TRAIL compared with the TRAIL-sensitive cell line, H460. PYCR1 knockdown in H1299 cells increased TRAIL sensitivity, increased the localization of death receptors (DRs) on the cell surface and activated Caspase-3/8. By contrast, overexpression of PYCR1 in H1299 cells decreased TRAIL sensitivity, reduced the distribution of DRs on the cell surface and suppressed the activation of Caspase-3/8. Taken together, these results suggested that PYCR1 promoted TRAIL resistance in the non-small cell lung cancer cell line, H1299, by preventing redistribution of DRs to the plasma membrane. This in turn inhibited TRAIL-mediated cell apoptosis by reducing the activation of Caspase-3/8.

Chemical Activation and Mechanical Sensitization of Piezo1 Enhance TRAIL-Mediated Apoptosis in Glioblastoma Cells.

Glioblastoma multiforme (GBM), the most common and aggressive type of primary brain tumor, has a mean survival of less than 15 months after standard treatment. Treatment with the current standard of care, temozolomide (TMZ), may be ineffective if damaged tumor cells undergo DNA repair or acquire mutations that inactivate transcription factor p53. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in multiple tumor types, while evading healthy cells, through a transcription-independent mechanism. GBM is particularly resistant to TRAIL, but studies have found that the mechanoreceptor Piezo1 can be activated under static conditions via Yoda1 agonist to induce TRAIL sensitization in other cancer cell lines. This study examines the effects and the mechanism of chemical and mechanical activation of Piezo1, via Yoda1 and fluid shear stress (FSS) stimulation, on TRAIL-mediated apoptosis in GBM cells. Here, we demonstrate that Yoda1 + TRAIL and FSS + TRAIL combination therapies significantly increase apoptosis in two GBM cell lines relative to controls. Further, cells known to be resistant to TMZ were found to have higher levels of Piezo1 expression and were more susceptible to TRAIL sensitization by Piezo1 activation. The combinatory Yoda1 + TRAIL treatment significantly decreased cell viability in TMZ-resistant GBM cells when compared to treatment with both low and high doses of TMZ. The results of this study suggest the potential of a highly specific and minimally invasive approach to overcome TMZ resistance in GBM by sensitizing cancer cells to TRAIL treatment via chemical or mechanical activation of Piezo1.

Decitabine enhances the tumoricidal potential of TRAIL via the epigenetic regulation of death receptor 4 in gastric cancer

Deoxyribonucleic acid (DNA) methyltransferase inhibitors, such as decitabine, have made great advances in cancer therapy as combinational drugs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has an obvious anti-tumor effect; however, some gastric cancer (GC) cells are resistant to TRAIL-induced cell death. This study sought to explore the synergistic anti-tumor effect of TRAIL and decitabine, and the potential synergetic mechanism. The cell growth inhibition effect was monitored by the IncuCyte ZOOM Live-Cell Analysis System, and cell viability was determined by Cell Counting Kit-8 assays. Apoptosis was detected by Annexin V/Propidium Iodide double staining. Death receptor 4 (DR4) was knocked down by ribonucleic acid (RNA) interference, and the effect of DR4 deletion on TRAIL sensitivity was analyzed. Methylation-specific polymerase chain reaction (PCR) was applied to determine the methylation status of DR4. The messenger RNA (mRNA) and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The expression of the DRs on the cell membrane surfaces was analyzed by flow cytometry. The combined use of decitabine and TRAIL synergistically inhibited cell growth in 2 TRAIL-resistant cell lines. Further, decitabine augmented TRAIL-induced apoptosis in a caspase-dependent manner. The co-application of decitabine and TRAIL facilitated the activation of caspase-7, -8, -9, and poly ADP-ribose polymerase (PARP). Notably, decitabine increased the expression of DR4 at the transcriptional and post-transcriptional levels. DR4 expression on the cell membrane surfaces was also upregulated after decitabine exposure. The depletion of DR4 by specific inhibitors attenuated TRAIL-induced apoptosis and weakened the synergistic effects of decitabine and TRAIL. In addition, DR4 gene presented methylation status in SNU-1 cells. The low mRNA and protein expression of DR4 were also detected in SNU-1 cells. Decitabine enhances the effect of TRAIL by inhibiting the growth and inducing the apoptosis of GC cells. This is achieved by the epigenetic modification of decitabine, which upregulates DR4. Decitabine may act as a sensitizing agent of TRAIL. The combined use of decitabine and TRAIL may provide a novel idea for GC treatment.

Rafoxanide sensitizes colorectal cancer cells to TRAIL-mediated apoptosis

Colorectal cancer (CRC) remains a leading causes of cancer-related death in the world, mainly due to the lack of effective treatment of advanced disease. TNF-related apoptosis-inducing ligand (TRAIL)-driven cell death, a crucial event in the control of tumor growth, selectively targets malignant rather than non-transformed cells. However, the fact that cancer cells, including CRC cells, are either intrinsically resistant or acquire resistance to TRAIL, represents a major hurdle to the use of TRAIL-based strategies in the clinic. Agents able to overcome CRC cell resistance to TRAIL have thus great therapeutic potential and many researchers are making efforts to identify TRAIL sensitizers. The anthelmintic drug rafoxanide has recently emerged as a potent anti-tumor molecule for different cancer types and we recently reported that rafoxanide restrained the proliferation of CRC cells, but not of normal colonic epithelial cells, both in vitro and in a preclinical model mimicking sporadic CRC. As these findings were linked with the induction of endoplasmic reticulum stress, a phenomenon involved in the regulation of various components of the TRAIL-driven apoptotic pathway, we sought to determine whether rafoxanide could restore the sensitivity of CRC cells to TRAIL. Our data show that rafoxanide acts as a selective TRAIL sensitizer in vitro and in a syngeneic experimental model of CRC, by decreasing the levels of c-FLIP and survivin, two key molecules conferring TRAIL resistance. Collectively, our data suggest that rafoxanide could potentially be deployed as an anti-cancer drug in the combinatorial approaches aimed at overcoming CRC cell resistance to TRAIL-based therapies.

Lewis glycosphingolipids as critical determinants of TRAIL sensitivity in cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces cancer cell death and contributes to tumor rejection by cytotoxic lymphocytes in cancer immunosurveillance and immunotherapy. TRAIL and TRAIL receptor agonists have garnered wide popularity as promising agents for cancer therapy. We previously demonstrated that the loss of fucosylation in cancer cells impairs TRAIL sensitivity; however, the precise structures of the fucosylated glycans that regulate TRAIL sensitivity and their carrier molecules remain elusive. Herein, we observed that Lewis glycans among various fucosylated glycans positively regulate TRAIL-induced cell death. Specifically, Lewis glycans on lacto/neolacto glycosphingolipids, but not glycoproteins including TRAIL receptors, enhanced TRAIL-induced formation of the cytosolic caspase 8 complex, without affecting the formation of the membranous receptor complex. Furthermore, type I Lewis glycan expression in colon cancer cell lines and patient-derived cancer organoids was positively correlated with TRAIL sensitivity. These findings provide novel insights into the regulatory mechanism of TRAIL-induced cell death and facilitate the identification of novel predictive biomarkers for TRAIL-related cancer therapies in future.

Licochalcone B induces DNA damage, cell cycle arrest, apoptosis, and enhances TRAIL sensitivity in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis. Traditional Chinese herbal medicines have been used to treat patients with cancer for many years in China. This study investigated the effects of licochalcone B (LCB), a flavonoid compound isolated from the root of Glycyrrhiza uralensis Fisch., on cell proliferation, DNA damage and TNF-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in HCC cells. Our results showed that LCB inhibited cell proliferation and induced DNA damage, cell cycle arrest and apoptosis. Treatment with LCB significantly inhibited the Akt/mTOR pathway and activated endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling pathway. Moreover, combined treatment with LCB and TRAIL yielded evident enhancements in the viability reduction and apoptosis. LCB upregulated death receptor 4 (DR4) and death receptor 5 (DR5) protein in a concentration- and time-dependent manner. The knockdown of DR5 significantly suppressed TRAIL-induced cleavage of PARP, which was enhanced by LCB. Treatment with an extracellular-regulated kinase (ERK) inhibitor (PD98059) or c-Jun N-terminal kinase (JNK) inhibitor (SP600125) markedly reduced the LCB-induced upregulation of DR5 expression and attenuated LCB-mediated TRAIL sensitization. In summary, LCB exhibits cytotoxic activity through modulation of the Akt/mTOR, ER stress and MAPK pathways in HCC cells and effectively enhances TRAIL sensitivity through the upregulation of DR5 expression in ERK- and JNK-dependent manner. Combination therapy with LCB and TRAIL may be an alternative treatment strategy for HCC.

Proscillaridin A Sensitizes Human Colon Cancer Cells to TRAIL-Induced Cell Death.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic cytokine that induces cancer cell death by binding to TRAIL receptors. Because of its selective cytotoxicity toward cancer cells, TRAIL therapeutics, such as recombinant TRAIL and agonistic antibodies targeting TRAIL receptors, have garnered attention as promising cancer treatment agents. However, many cancer cells acquire resistance to TRAIL-induced cell death. To overcome this issue, we searched for agents to sensitize cancer cells to TRAIL-induced cell death by screening a small-molecule chemical library consisting of diverse compounds. We identified a cardiac glycoside, proscillaridin A, as the most effective TRAIL sensitizer in colon cancer cells. Proscillaridin A synergistically enhanced TRAIL-induced cell death in TRAIL-sensitive and -resistant colon cancer cells. Additionally, proscillaridin A enhanced cell death in cells treated with TRAIL and TRAIL sensitizer, the second mitochondria-derived activator of caspase mimetic. Proscillaridin A upregulated TRAIL receptor expression, while downregulating the levels of the anti-cell death molecules, cellular FADD-like IL-1β converting enzyme-like inhibitor protein and Mcl1, in a cell type-dependent manner. Furthermore, proscillaridin A enhanced TRAIL-induced cell death partly via O-glycosylation. Taken together, our findings suggest that proscillaridin A is a promising agent that enhances the anti-cancer efficacy of TRAIL therapeutics.

EV-T synergizes with AZD5582 to overcome TRAIL resistance through concomitant suppression of cFLIP, MCL-1, and IAPs in hepatocarcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy, and its effective treatment has been hampered by drug resistance. Extracellular vesicle (EV) delivery of TNF-related apoptosis-inducing ligand (TRAIL) (EV-T) was demonstrated to be superior to recombinant TRAIL (rTRAIL) for cancer treatment previously. And AZD5582, a potent antagonist of inhibitors of apoptosis proteins (IAPs) can potentiate apoptosis-based cancer therapies. However, the combination of EV-T and AZD5582 has never been examined for their possible apoptosis inducing synergism in cancers. In this study, we proposed and tested the combination of EV-T and AZD5582 as a potential novel therapy for effective treatment of HCC. Two HCC lines Huh7 and HepG2 that are both resistant to rTRAIL were examined. The results confirmed that AZD5582 and EV-T are synergistic for apoptosis induction in some cancer lines including Huh7 and HepG2 while sparing normal cells. More importantly, this study revealed that TRAIL sensitization by AZD5582 is mediated through the concomitant suppression of anti-apoptotic factors including cFLIP, MCL-1, and IAPs (XIAP, Survivin and cIAP-1). Particularly the downregulation of cFLIP and IAP's appeared to be essential and necessary for the synergism between AZD5582 and TRAIL. In vivo, we first time demonstrated that the combined therapy with low doses of AZD5582 and EV-Ts triggered drastically enhanced apoptosis leading to the complete eradication of Huh7 tumor development without any apparent adverse side effects examined. We thus have unraveled the important molecular mechanism underlying TRAIL sensitization by AZD5582, rationalizing the next development of a combination therapy with AZD5582 and EV-T for HCC treatment. KEY MESSAGES: It confirmed the TRAIL sensitization by AZD5582, a potent antagonist of IAPs in hepatocarcinoma. It revealed that the sensitization is via the concomitant suppression of antiapoptotic factors including cFLIP, MCL-1, and IAPs. The downregulation of cFLIP and IAPs like Survivin appeared to be essential and necessary for the synergism between AZD5582 and nanosomal TRAIL. In vivo the combined therapy with AZD5582 and nanosomal TRAIL led to complete eradication of hepatocarcinoma tumors. This study has rationalized the next development of a combination therapy with AZD5582 and nanosomal TRAIL for cancer treatment.

Impact of Extracellular pH on Apoptotic and Non-Apoptotic TRAIL-Induced Signaling in Pancreatic Ductal Adenocarcinoma Cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an important mediator of tumor immune surveillance. In addition, its potential to kill cancer cells without harming healthy cells led to the development of TRAIL receptor agonists, which however did not show the desired effects in clinical trials. This is caused mainly by apoptosis resistance mechanisms operating in primary cancer cells. Meanwhile, it has been realized that in addition to cell death, TRAIL also induces non-apoptotic pro-inflammatory pathways that may enhance tumor malignancy. Due to its late detection and resistance to current therapeutic options, pancreatic ductal adenocarcinoma (PDAC) is still one of the deadliest types of cancer worldwide. A dysregulated pH microenvironment contributes to PDAC development, in which the cancer cells become highly dependent on to maintain their metabolism. The impact of extracellular pH (pHe) on TRAIL-induced signaling in PDAC cells is poorly understood so far. To close this gap, we analyzed the effects of acidic and alkaline pHe, both in short-term and long-term settings, on apoptotic and non-apoptotic TRAIL-induced signaling. We found that acidic and alkaline pHe differentially impact TRAIL-induced responses, and in addition, the duration of the pHe exposition also represents an important parameter. Thus, adaptation to acidic pHe increases TRAIL sensitivity in two different PDAC cell lines, Colo357 and Panc1, one already TRAIL-sensitive and the other TRAIL-resistant, respectively. However, the latter became highly TRAIL-sensitive only by concomitant inhibition of Bcl-xL. None of these effects was observed under other pHe conditions studied. Both TRAIL-induced non-apoptotic signaling pathways, as well as constitutively expressed anti-apoptotic proteins, were regulated by acidic pHe. Whereas the non-apoptotic pathways were differently affected in Colo357 than in Panc1 cells, the impact on the anti-apoptotic protein levels was similar in both cell lines. In Panc1 cells, adaptation to either acidic or alkaline pHe blocked the activation of the most of TRAIL-induced non-apoptotic pathways. Interestingly, under these conditions, significant downregulation of the plasma membrane levels of TRAIL-R1 and TRAIL-R2 was observed. Summing up, extracellular pH influences PDAC cells’ response to TRAIL with acidic pHe adaptation, showing the ability to strongly increase TRAIL sensitivity and in addition to inhibit TRAIL-induced pro-inflammatory signaling.

Embelin downregulated cFLIP in breast cancer cell lines facilitate anti-tumor effect of IL-1\u03b2-stimulated human umbilical cord mesenchymal stem cells

Breast cancer is the leading cause of cancer-related death for women. In breast cancer treatment, targeted therapy would be more effective and less harmful than radiotherapy or systemic chemotherapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in cancer cells but not in normal cells. Mesenchymal stem cells have shown great therapeutic potential in cancer therapy owing to their ability of homing to tumor sites and secreting many kinds of anti-tumor proteins including TRAIL. In this study, we found that IL-1β-stimulated human umbilical cord-derived mesenchymal stem cells (hUCMSCs) enhance the expression of membrane-bound and soluble TRAIL. Cellular FADD-like IL-1β-converting enzyme inhibitory protein (cFLIP) is an important regulator in TRAIL-mediated apoptosis and relates to TRAIL resistance in cancer cells. Previous studies have shown that embelin, which is extracted from Embelia ribes, can increase the TRAIL sensitivity of cancer cells by reducing cFLIP expression. Here we have demonstrated that cFLIPL is correlated with TRAIL-resistance and that embelin effectively downregulates cFLIPL in breast cancer cells. Moreover, co-culture of IL-1β-stimulated hUCMSCs with embelin-treated breast cancer cells could effectively induce apoptosis in breast cancer cells. The combined effects of embelin and IL-1β-stimulated hUCMSCs may provide a new therapeutic strategy for breast cancer therapy.

TRAIL-R3 Expression on Myeloid Leukemic Blasts Is Related to a Shortened Overall Survival.

Since chemotherapy and transplantation can cure only around 35% of patients with acute myeloid leukemia (AML), there is still need for complementary and targeted treatment modalities. One of them could be the use of TNF-related apoptosis-inducing ligand (TRAIL). TRAIL is expressed by effector T cells and induces apoptosis via the death receptor intrinsic pathway. Activation of this pathway in addition to the mitochondrial pathway (by chemotherapy) has synergistic effects in vitro. In human, 4 membrane bound receptors have been identified: two of them (R1 and R2) contain a functional death domain and are capable of starting the apoptotic cascade, and two others (R3 and R4) lack a functional death domain and function as decoy receptors. Most normal cells express R3 and R4, where many tumor cells express R1 and R2. This makes soluble recombinant TRAIL an attractive candidate for targeted therapy; phase 1 clinical studies for solid tumors are launched. Until now, sparse data on TRAIL sensitivity of myeloid leukemic cells have demonstrated low TRAIL sensitivity. We investigated blood and bone-marrow samples of 113 patients with AML for TRAIL receptor expression by flow-cytometry. In contrast with published data, we found presumably (pro-apoptotic) R1 and R2 expression (mean percentage positive cells 16% and 34%, range 0–79% and 0–97% respectively) versus R3 and R4 expression (mean 9% and 10%, range 0–71% an 0–45%) indicating a TRAIL sensitive profile for myeloid blasts. Surprisingly, the expression of the anti-apoptotic R3 strongly correlated to survival. Expression of > 25% blasts positive for R3 resulted in shortened overall survival (p=0.0051), see figure 1. In multivariate analysis R3 expression remained a significant prognostic factor next to cytogenetics (p=0.03 and p= 0.015 respectively). In vitro studies on 4 myeloid leukemic cell lines confirmed TRAIL sensitivity in cell lines that expressed R1 and R2. Furthermore, simultaneous expression of R3 clearly reduced the amount of apoptosis, suggesting that TRAIL effects are inhibited by binding to R3. TRAIL induction studies on fresh leukemic samples are currently underway in our laboratory.In conclusion, our data suggest that, in contrast to earlier reports, there might be a role for TRAIL in apoptosis induction of AML blasts. R3 expression is a strong predictor for overall survival and modulation of R3 might yield additional new therapeutic options for AML patients. [Display omitted]

Traditional Chinese medicines and their active ingredients sensitize cancer cells to TRAIL-induced apoptosis.

The rapidly developing resistance of cancers to chemotherapy agents and the severe cytotoxicity of such agents to normal cells are major stumbling blocks in current cancer treatments. Most current chemotherapy agents have significant cytotoxicity, which leads to devastating adverse effects and results in a substandard quality of life, including increased daily morbidity and premature mortality. The death receptor of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can sidestep p53-dependent pathways to induce tumor cell apoptosis without damaging most normal cells. However, various cancer cells can develop resistance to TRAIL-induced apoptosis via different pathways. Therefore, it is critical to find an efficient TRAIL sensitizer to reverse the resistance of tumor cells to TRAIL, and to reinforce TRAIL's ability to induce tumor cell apoptosis. In recent years, traditional Chinese medicines and their active ingredients have shown great potential to trigger apoptotic cell death in TRAIL-resistant cancer cell lines. This review aims to collate information about Chinese medicines that can effectively reverse the resistance of tumor cells to TRAIL and enhance TRAIL's ability to induce apoptosis. We explore the therapeutic potential of TRAIL and provide new ideas for the development of TRAIL therapy and the generation of new anti-cancer drugs for human cancer treatment. This study involved an extensive review of studies obtained from literature searches of electronic databases such as Google Scholar and PubMed. "TRAIL sensitize" and "Chinese medicine" were the search keywords. We then isolated newly published studies on the mechanisms of TRAIL-induced apoptosis. The name of each plant was validated using certified databases such as The Plant List. This study indicates that TRAIL can be combined with different Chinese medicine components through intrinsic or extrinsic pathways to promote cancer cell apoptosis. It also demonstrates that the active ingredients of traditional Chinese medicines enhance the sensitivity of cancer cells to TRAIL-mediated apoptosis. This provides useful information regarding traditional Chinese medicine treatment, the development of TRAIL-based therapies, and the treatment of cancer.

Taxanes Sensitize Prostate Cancer Cells to TRAIL-Induced Apoptotic Synergy via Endoplasmic Reticulum Stress.

Docetaxel and cabazitaxel are guideline-chemotherapy treatments for metastatic castration-resistant prostate cancer (mCRPC), which comprises the majority of prostate cancer deaths. TNF-related apoptosis inducing ligand (TRAIL) is an anticancer agent that is selectively cytotoxic to cancer cells; however, many human cancers are resistant to TRAIL. In this study, we sensitized androgen-independent and TRAIL-resistant prostate cancer cells to TRAIL-mediated apoptosis via taxane therapy and examined the mechanism of sensitization. DU145 and PC3 cells displayed no significant reduction in cell viability when treated with soluble TRAIL, docetaxel, or cabazitaxel alone indicating that both cell lines are resistant to TRAIL and taxanes individually. Taxane and TRAIL combination synergistically amplified apoptosis strongly suggesting that taxanes sensitize prostate cancer cells to TRAIL. A Jun N-terminal kinases (JNK) inhibitor inhibited apoptosis in treated cells and significantly reduced death receptor expression indicating JNK activation by ER stress sensitizes PCa cells to TRAIL-induced apoptosis by upregulating DR4/DR5 expression. In addition, suppression of C/EBP homologous protein (CHOP) reduced TRAIL sensitization in both cell lines indicating that ER stress-related apoptosis is mediated, in part, by CHOP. Cytochrome c knockdown showed a significant decrease in sensitivity in PC3 cells, but not in Bax-deficient DU145 cells. A computational model was used to simulate apoptosis for cells treated with taxane and TRAIL therapy as demonstrated in in vitro experiments. Pretreatment with taxanes sensitized cells to apoptosis induced by TRAIL-mediated apoptosis, demonstrating that combining TRAIL with ER stress inducers is a promising therapy to reverse TRAIL resistance to treat mCRPC.

Abstract A17: Targeting PI3K/Akt-mediated MADD phosphorylation improves TRAIL sensitivity in anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer is an extremely hard-to-treat malignancy due to its aggressiveness, metastasis and inherent therapeutic resistance. Despite using all available armamentaria, the median survival of ATC patients remains dismal. In this regard, TNF-related apoptosis-inducing ligand (TRAIL) is known to induce cancer-specific apoptosis without affecting nontumor cells. However, development of resistance to TRAIL hampers its clinical use. One of the mechanisms of TRAIL resistance is mediated through phosphorylation of Map kinase Activating Death Domain-containing protein (MADD) by Akt. In light of this, preventing MADD phosphorylation might be a rational approach to overcome TRAIL resistance. To test this, we examined MADD expression and TRAIL sensitivity by Western blotting and active caspase-3 staining in several ATC cell lines harboring mutations in cancer driver genes including BRAF, Akt, and PTEN. MADD phosphorylation was inhibited by introducing site-directed mutations in Akt phosphorylation sites of MADD and exogenously expressing the mutant constructs to determine the effect on TRAIL-induced apoptosis. Our results showed that MADD was differentially overexpressed in all ATC cells compared to the normal thyroid tissues irrespective of their mutational landscape. Further, exogenous expression of non-phosphorylatable MADD resulted in a significantly increased TRAIL-induced apoptosis accompanied by the activation of both intrinsic and extrinsic apoptotic pathways. Thus, this study establishes a concept of prevention of MADD phosphorylation to improve TRAIL susceptibility of ATC cells as it can yield similar results as that of blocking PI3k/Akt pathway without any off-target effects. Further studies are warranted to explore specific inhibitors to target MADD phosphorylation. Note: This abstract was not presented at the conference. Citation Format: Shikha Saini, Aditi Mathur, Fei Yue, Ajay Maker, Bellur S. Prabhakar. Targeting PI3K/Akt-mediated MADD phosphorylation improves TRAIL sensitivity in anaplastic thyroid cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A17.

Cytochalasins from an endophytic fungus Phoma multirostrata XJ-2-1 with cell cycle arrest and TRAIL-resistance-overcoming activities

Nine new (1–9) and four known (10–13) [13]cytochalasins, along with three known 24-oxa[14]cytochalasins (14–16), were isolated from the culture of Phoma multirostrata XJ-2-1, an endophytic fungus obtained from the fibrous root of Parasenecio albus. Their structures were elucidated by interpretation of the nuclear magnetic resonance (NMR) and high-resolution electrospray ionization mass spectroscopy (HRESIMS). The absolute configurations were assigned by single-crystal X-ray crystallography, modified Mosher′s method, and by analysis of their experimental electronic circular dichroism (ECD) spectra. Compound 6 could induce cell cycle arrest at G2-phase in CT26 and A549 cells, and displayed moderate cytotoxicity against CT26 and A549 cell lines with IC50 values of 6.03 and 5.04 μM, respectively. Co-treatment of 7–9, 13 and 16 with tumor necrosis factor related apoptosis inducing ligand (TRAIL) could significantly decrease the cell viability of A549, which revealed that cytochalasins could possibly be a new group of TRAIL sensitizers in lung cancer therapy.

Upregulation of DR5 and Downregulation of Survivin by IITZ-01, Lysosomotropic Autophagy Inhibitor, Potentiates TRAIL-Mediated Apoptosis in Renal Cancer Cells via Ubiquitin-Proteasome Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively is able to increase apoptosis in cancer cells as agent with minimum toxicity to noncancerous cells. However, all cancer cells are not sensitive to TRAIL-induced apoptosis. In this study, we showed the sub-lethal concentrations of a lysosomotropic autophagy inhibitor, IITZ-01, sensitizes cancer cells (renal, lung, and breast carcinoma) to TRAIL-induced apoptosis through DR5 upregulation and survivin downregulation through ubiquitin-proteasome pathway. Knockdown of DR5 or overexpression of survivin inhibited combined treatment with IITZ-01 and TRAIL-induced apoptosis. IITZ-01 downregulated protein expression of Cbl, ubiquitin E3 ligase, and decreased expression level of Cbl markedly led to increase DR5 protein expression and TRAIL sensitivity. Moreover, IITZ-01 decreased expression level of survivin protein via downregulation of deubiquitinase ubiquitin-specific protease 9X (USP9X) expression. Taken together, these results provide the first evidence that IITZ-01 enhances TRAIL-mediated apoptosis through DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation in renal carcinoma cells.

Romo1 Inhibition Induces TRAIL-Mediated Apoptosis in Colorectal Cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is known to behave as an attractive anti-cancer agent in various cancers. Despite its promise TRAIL has limitations such as short half-life and rapid development of resistance. In this regard, approaches to sensitizers of TRAIL that can overcome the limitations of TRAIL are necessary. However, the molecular targets and mechanisms underlying sensitization to TRAIL-induced apoptosis are not fully understood. Here, we propose that reactive oxygen species modulator-1 (Romo1) as an attractive sensitizer of TRAIL. Romo1 is a mitochondrial inner membrane channel protein that controls reactive oxygen species (ROS) production, and its expression is highly upregulated in various cancers, including colorectal cancer. In the present study, we demonstrated that Romo1 inhibition significantly increased TRAIL-induced apoptosis of colorectal cancer cells, but not of normal colon cells. The combined effect of TRAIL and Romo1 inhibition was correlated with the activation of mitochondrial apoptosis pathways. Romo1 silencing elevated the protein levels of BCL-2-associated X protein (Bax) by downregulating the ubiquitin proteasome system (UPS). Romo1 inhibition downregulated the interaction between Bax and Parkin. Furthermore, Romo1 knockdown triggered the mitochondrial dysfunction and ROS generation. We validated the effect of combination in tumor xenograft model in vivo. In conclusion, our study demonstrates that Romo1 inhibition induces TRAIL-mediated apoptosis by identifying the novel mechanism associated with the Bax/Parkin interaction. We suggest that targeting of Romo1 is essential for the treatment of colorectal cancer and may be a new therapeutic approach in the future and contribute to the drug discovery.

Andrographolide sensitizes human renal carcinoma cells to TRAIL‑induced apoptosis through upregulation of death receptor 4

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

Tristetraprolin Posttranscriptionally Downregulates TRAIL Death Receptors.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has attracted attention as a potential candidate for cancer therapy. However, many primary cancers are resistant to TRAIL, even when combined with standard chemotherapy. The mechanism of TRAIL resistance in cancer cells has not been fully elucidated. The TRAIL death receptor (DR) 3′-untranslated region (3′-UTR) is reported to contain AU-rich elements (AREs) that are important for regulating DR mRNA stability. However, the mechanisms by which DR mRNA stability is determined by its 3′-UTR are unknown. We demonstrate that tristetraprolin (TTP), an ARE-binding protein, has a critical function of regulating DR mRNA stability. DR4 mRNA contains three AREs and DR5 mRNA contains four AREs in 3′-UTR. TTP bound to all three AREs in DR4 and ARE3 in DR5 and enhanced decay of DR4/5 mRNA. TTP overexpression in colon cancer cells changed the TRAIL-sensitive cancer cells to TRAIL-resistant cells, and down-regulation of TTP increased TRAIL sensitivity via DR4/5 expression. Therefore, this study provides a molecular mechanism for enhanced levels of TRAIL DRs in cancer cells and a biological basis for posttranscriptional modification of TRAIL DRs. In addition, TTP status might be a biomarker for predicting TRAIL response when a TRAIL-based treatment is used for cancer.