The Effect of Xevinapant Combined with Ionizing Radiation on HNSCC and Normal Tissue Cells and the Impact of Xevinapant on Its Targeted Proteins cIAP1 and XIAP.

The poor prognosis of HNSCC is partly due to treatment resistance. The SMAC mimetic Xevinapant is a promising new approach to targeted cancer therapy. Xevinapant inhibits cIAP1/2 and XIAP, leading to apoptosis, necroptosis and inhibition of prosurvival signaling. Combining Xevinapant with IR could improve therapeutic potential. The effect of Xevinapant in combination with IR on HNSCC and healthy tissue cells was investigated. Cell growth, cell death, clonogenic survival and DNA double-strand breaks (DSBs) were studied, and intracellular cIAP1 and XIAP levels were evaluated. Xevinapant had cytostatic and cytotoxic, as well as radiosensitizing, effects on the malignant cells, while healthy tissue cells were less affected. Apoptotic and necrotic cell death was particularly affected, but the increase in residual DSBs and the reduced survival implied an additional effect of Xevinapant on DNA damage repair and other cell inactivation mechanisms. cIAP1 and XIAP levels varied for each cell line and were affected by Xevinapant and IR treatment. There was an association between higher IAP levels and increased cell death. Xevinapant appears to be a potent new drug for HNSCC therapy, especially in combination with IR. IAP levels could be an indicator for impaired DNA damage repair and increased susceptibility to cellular stress.

Cell-to-cell variability in inducible Caspase9-mediated cell death.

iCasp9 suicide gene has been widely used as a promising killing strategy in various cell therapies. However, different cells show significant heterogeneity in response to apoptosis inducer, posing challenges in clinical applications of killing strategy. The cause of the heterogeneity remains elusive so far. Here, by simultaneously monitoring the dynamics of iCasp9 dimerization, Caspase3 activation, and cell fate in single cells, we found that the heterogeneity was mainly due to cell-to-cell variability in initial iCasp9 expression and XIAP/Caspase3 ratio. Moreover, multiple-round drugging cannot increase the killing efficiency. Instead, it will place selective pressure on protein levels, especially on the level of initial iCasp9, leading to drug resistance. We further show this resistance can be largely eliminated by combinatorial drugging with XIAP inhibitor at the end, but not at the beginning, of the multiple-round treatments. Our results unveil the source of cell fate heterogeneity and drug resistance in iCasp9-mediated cell death, which may enlighten better therapeutic strategies for optimized killing.

Embelin: A novel XIAP inhibitor for the prevention and treatment of chronic diseases.

Chronic diseases are a serious health concern worldwide, especially in the elderly population. Most chronic diseases like cancer, cardiovascular ailments, neurodegenerative disorders, and autoimmune diseases are caused due to the abnormal functioning of multiple signaling pathways that give rise to critical anomalies in the body. Although a lot of advanced therapies are available, these have failed to entirely cure the disease due to their less efficacy. Apart from this, they have been shown to manifest disturbing side effects which hamper the patient's quality of life to the extreme. Since the last few decades, extensive studies have been done on natural herbs due to their excellent medicinal benefits. Components present in natural herbs target multiple signaling pathways involved in diseases and therefore hold high potential in the prevention and treatment of various chronic diseases. Embelin, a benzoquinone, is one such agent isolated from Embelia ribes, which has shown excellent biological activities toward several chronic ailments by upregulating a number of antioxidant enzymes (e.g., SOD, CAT, GSH, etc.), inhibiting anti-apoptotic genes (e.g., TRAIL, XIAP, survivin, etc.), modulating transcription factors (e.g., NF-κB, STAT3, etc.) blocking inflammatory biomarkers (e.g., NO, IL-1ß, IL-6, TNF-α, etc.), monitoring cell cycle synchronizing genes (e.g., p53, cyclins, CDKs, etc.), and so forth. Several preclinical studies have confirmed its excellent therapeutic activities against malicious diseases like cancer, obesity, heart diseases, Alzheimer's, and so forth. This review presents an overview of embelin, its therapeutic prospective, and the molecular targets in different chronic diseases.

Induction of apoptosis, cytotoxicity and radiosensitization by novel 3,4-dihydroquinazolinone derivatives.

Twenty new quinazolinone derivatives bearing a piperonyl moiety were designed and synthesized. The structures of the target compounds were in agreement with the microanalytical and spectral data. Compounds 4-10, 13, 14 and 17-27 were screened for their cytotoxic activity against HepG-2 and MCF-7 cancer cell lines. The target compounds showed IC50 in the range of 2.46-36.85 µM and 3.87-88.93 µM for HepG-2 and MCF-7, respectively. The promising compounds 7, 19, 26 and 27 were selected to measure their EGFR inhibitory activity. The IC50 values of the promising compounds were in the range of 146.9-1032.7 nM for EGFR in reference to Erlotinib (IC50 = 96.6 nM). In further studies on compounds 7, 19, 26 and 27 using HepG-2 cell line, there was significant overexpression of p21 and downregulation of two members of IAPs protein family; Survivin and XIAP, relative to their controls. Annexin V-FITC and caspase-3 analyses have established a significant increase in early apoptosis. Moreover, the four selected compounds have impaired cell proliferation by cell cycle arrest at the G2/M phase compared to their respective control. Considering radiotherapy as the primary treatment for many types of solid tumors, the radiosensitizing abilities of compounds 7, 19, 26 and 27 were measured against HepG-2 and MCF-7 cell lines combined with a single dose of 8 Gy gamma radiation. Measurement of the IC50 of the promising compounds after irradiation revealed their ability to sensitize the cells to the lethal effect of gamma irradiation (IC50 = 1.56-4.32 µM and 3.06-5.93 µM for HepG-2 and MCF-7 cells, respectively). Molecular docking was performed to gain insights into the ligand-binding interactions of 7, 19, 26 and 27 inside the EGFR binding sites and revealed their essential interactions, explaining their good activity towards EGFR.

Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Embelin potentiates venetoclax-induced apoptosis in acute myeloid leukemia cells.

Acute myeloid leukemia (AML) belongs to a group of hematological cancer whose relapse cases are often associated with chemoresistance that impairs treatment success and contributes to a poor outcome. For this reason, there is an urgent need for the development of new therapeutic strategies. Herein, we explore the combination of venetoclax, a BCL2 inhibitor, and embelin, an XIAP inhibitor, in the AML cell lines. Combinatory treatment of venetoclax and embelin potentiated cytotoxic effects of these drugs, demonstrating that both in combination present lower IC50 values than single treatment of either venetoclax or embelin alone in both cell lines analyzed. The combinatory treatment further increased the apoptosis-inducing properties of both compounds. Computer simulations suggest that embelin binds to both BIR2 and BIR3 domains of XIAP, reinforcing this inhibitory apoptosis protein as an embelin target. Although all AML cell lines presented similar basal levels of XIAP, the combinatory treatment effectively inhibited XIAP expression in OCI-AML3 cells. In conclusion, the inhibition of both apoptosis inhibitory players, BCL2 and XIAP, by venetoclax and embelin, respectively, potentiated their cytotoxic effects in AML cell lines.

X-linked inhibitor of apoptosis protein (XIAP) inhibition in systemic sclerosis (SSc).

OBJECTIVE: X-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc). METHODS: The expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice. RESULTS: The expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-ß) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/ß-catenin signalling. Inactivation of XIAP reduces binding of ß-catenin to TCF to in a TLE-dependent manner to block WNT/ß-catenin-dependent transcription. CONCLUSIONS: Our data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-ß/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/ß-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.

Targeting Cutaneous T-Cell Lymphoma Cells by Ingenol Mebutate (PEP005) Correlates with PKCδ Activation, ROS Induction as Well as Downregulation of XIAP and c-FLIP.

New therapeutic strategies are needed for cutaneous T-cell lymphoma (CTCL), and the plant extract ingenol mebutate (PEP005) may be considered. PEP005 has been approved for actinic keratosis, and proapoptotic activities were described in different cancer cells. Here, we aimed to investigate its efficacy in four CTCL cell lines and its mode of action. While HuT-78 and HH responded with induced apoptosis as well as with loss of cell viability and cell proliferation, MyLa and SeAx remained resistant. Interestingly, both sensitive and resistant cells showed caspase-8 activation and enhanced levels of reactive oxygen species (ROS), while final caspase-3 activation was restricted to sensitive cells. Apoptosis induction was prevented by the caspase inhibitor QVD-Oph as well as by the antioxidant vitamin E. Caspase activation by PEP005 may be explained to some extent by the downregulation of the caspase antagonistic proteins c-FLIP and XIAP in sensitive cells, whereas both proteins were strongly expressed in resistant cells. Finally, PEP005 resulted in the activation of proapoptotic PKCδ, and the PKC inhibitor bisindolylmaleimide I reduced apoptosis, caspase-3 processing and ROS production, as well as restored cell viability. In conclusion, PKCδ appeared as a central player in apoptosis regulation in CTCL cells, also suggesting its therapeutic targeting.

Structure based pharmacophore modeling, virtual screening, molecular docking and ADMET approaches for identification of natural anti-cancer agents targeting XIAP protein.

X-linked inhibitor of apoptosis protein (XIAP) is a member of inhibitor of apoptosis protein (IAP) family responsible for neutralizing the caspases-3, caspases-7, and caspases-9. Overexpression of the protein decreased the apoptosis process in the cell and resulting development of cancer. Different types of XIAP antagonists are generally used to repair the defective apoptosis process that can eliminate carcinoma from living bodies. The chemically synthesis compounds discovered till now as XIAP inhibitors exhibiting side effects, which is making difficulties during the treatment of chemotherapy. So, the study has design to identifying new natural compounds that are able to induce apoptosis by freeing up caspases and will be low toxic. To identify natural compound, a structure-based pharmacophore model to the protein active site cavity was generated following by virtual screening, molecular docking and molecular dynamics (MD) simulation. Initially, seven hit compounds were retrieved and based on molecular docking approach four compounds has chosen for further evaluation. To confirm stability of the selected drug candidate to the target protein the MD simulation approach were employed, which confirmed stability of the three compounds. Based on the finding, three newly obtained compounds namely Caucasicoside A (ZINC77257307), Polygalaxanthone III (ZINC247950187), and MCULE-9896837409 (ZINC107434573) may serve as lead compounds to fight against the treatment of XIAP related cancer, although further evaluation through wet lab is necessary to measure the efficacy of the compounds.

Discovery of N-(3,4-Dimethylphenyl)-4-(4-isobutyrylphenyl)-2,3,3a,4,5,9b-hexahydrofuro[3,2-c]quinoline-8-sulfonamide as a Potent Dual MDM2/XIAP Inhibitor.

Murine double minute 2 (MDM2) and X-linked inhibitor of apoptosis protein (XIAP) are important cell survival proteins in tumor cells. As a dual MDM2/XIAP inhibitor reported previously, compound MX69 has low potency with an IC50 value of 7.5 µM against an acute lymphoblastic leukemia cell line EU-1. Herein, we report the structural optimization based on the MX69 scaffold, leading to the discovery of a 25-fold more potent analogue 14 (IC50 = 0.3 µM against EU-1). We demonstrate that 14 maintains its mode of action by dual targeting of MDM2 and XIAP through inducing MDM2 protein degradation and inhibiting XIAP mRNA translation, respectively, which resulted in cancer cell growth inhibition and cell death. The results strongly suggest that the scaffold based on 14 is promising for further optimization to develop a new therapeutic agent for leukemia and possibly other cancers where MDM2 and XIAP are dysregulated.

MicroRNA-215 Regulates the Apoptosis of HCT116 Colon Cancer Cells by Inhibiting X-Linked Inhibitor of Apoptosis Protein.

Background: X-linked inhibitor of apoptosis protein (XIAP) is the strongest member of the family of inhibitor of apoptosis protein. Studies found that the expression of XIAP in colon cancer tissue was significantly higher than that in adjacent tissues. Studies have shown that the expression of microRNA-215 (miR-215) was significantly lower than that of the adjacent tissues. This study investigated whether dysregulated miR-215 and XIAP play important roles in colon cancer cell apoptosis and the incidence of colon cancer. Materials and Methods: Forty-two patients with colorectal cancer (CRC) diagnosed and treated in the authors' hospital were selected. Human CRC cell line HCT116 and normal colonic mucosal epithelial cells (CMECs) were used. Luciferase reporter gene vector was constructed and dual-luciferase reporter gene assay was performed. HCT116 cells were cultured in vitro and divided into five groups: mimic normal control (NC) group, miR-215 mimic group, si-NC group, si-XIAP group, and miR-215 mimic + si-XIAP group. Western blot and polymerase chain reaction were conducted to examine XIAP and caspase-3. Apoptosis was detected by flow cytometry and cell proliferation was detected by cell counting kit-8 assay. Results: Compared with the adjacent tissues, the expression of miR-215 in colon cancer tissue was significantly lower, whereas the expression of XIAP in colon cancer tissue was significantly higher. The apoptosis rate and miR-215 expression level of HCT116 cells were lower than that of normal CMECs, whereas XIAP expression was significantly higher than that in normal colon mucosa epithelial cells. MiR-215 targeted the 3'-untranslated regions of XIAP and inhibited its expression. Overexpressing miR-215 and (or) silencing XIAP expression could significantly enhance the activity of caspase-9 and caspase-3, and promote the apoptosis of HCT116 cells. Conclusion: MiR-215 inhibited the expression of XIAP and promoted the apoptosis of HCT116 cells.

Targeting X chromosome-linked inhibitor of apoptosis protein in mucoepidermoid carcinoma of the head and neck: A novel therapeutic strategy using nitidine chloride.

Nitidine chloride (NC) was recently reported to exhibit a wide range of pharmacological properties for several diseases, including cancer. Here we report for the first time that NC is a potential therapeutic agent for mucoepidermoid carcinoma (MEC) occurring in the head and neck because it suppresses X chromosome-linked inhibitor of apoptosis protein (XIAP) in human MEC in vitro and in vivo. The antitumor effects of NC were evaluated by trypan blue exclusion assay, western blotting, live/dead assay, 4',6-diamidino-2-phenylindole (DAPI) staining, human apoptosis antibody array, immunofluorescence staining, immunohistochemistry, small interfering RNA assay, transient transfection of XIAP overexpression vector, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and histopathological examination of organs. NC inhibited cell viability and induced caspase-dependent apoptosis in vitro. A human apoptosis antibody array assay showed that XIAP is suppressed by NC treatment. XIAP was overexpressed in oral squamous cell carcinoma (OSCC) tissues that arose from the head and neck, and high XIAP expression was correlated with poor prognosis in OSCC patients. XIAP depletion significantly increased apoptosis, and ectopic XIAP overexpression attenuated the apoptosis induced by NC treatment. NC suppressed tumor growth in vivo at a dosage of 5 mg/kg/day. The number of TUNEL-positive cells increased and the protein expression of XIAP was consistently downregulated in NC-treated tumor tissues. In addition, NC caused no histopathological changes in the liver or kidney. These findings provide new insights into the mechanism of action underlying the anticancer effects of NC and demonstrate that NC is a promising therapeutic agent for the treatment of human MEC of the head and neck. KEY MESSAGES: • Nitidine chloride induces caspase-dependent apoptosis in MEC of the head and neck. • High XIAP expression correlates with poor prognosis of OSCC patients. • Nitidine chloride suppresses tumor growth in vivo without any systemic toxicities. • Targeting XIAP is a novel chemotherapeutic strategy for MEC of the head and neck.

Stability and Cell Permeability of Sulfonyl Fluorides in the Design of Lys-Covalent Antagonists of Protein-Protein Interactions.

Recently we reported on aryl-fluorosulfates as possible stable and effective electrophiles for the design of lysine covalent, cell permeable antagonists of protein-protein interactions (PPIs). Here we revisit the use of aryl-sulfonyl fluorides as Lys-targeting moieties, incorporating these electrophiles in XIAP (X-linked inhibitor of apoptosis protein) targeting agents. We evaluated stability in buffer and reactivity with Lys311 of XIAP of various aryl-sulfonyl fluorides using biochemical and biophysical approaches, including displacement assays, mass spectrometry, SDS gel electrophoresis, and denaturation thermal shift measurements. To assess whether these modified electrophilic "warheads" can also react with Tyr, we repeated these evaluations with a Lys311Tyr XIAP mutant. Using a direct cellular assay, we could demonstrate that selected agents are cell permeable and interact covalently with their intended target in cell. These results suggest that certain substituted aryl-sulfonyl fluorides can be useful Lys- or Tyr-targeting electrophiles for the design of covalent pharmacological tools or even future therapeutics targeting protein-protein interactions.

Tumor necrosis factor­related apoptosis­inducing ligand as a therapeutic option in urothelial cancer cells with acquired resistance against first­line chemotherapy.

Patients with urothelial carcinoma frequently fail to respond to first­line chemotherapy using cisplatin and gemcitabine due to development of resistant tumor cells. The aim of the present study was to investigate whether an alternative treatment with tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) that induces tumor cell death via the extrinsic apoptotic pathway may be effective against chemotherapy­resistant urothelial cancer cell lines. The viability of the urothelial cancer cell line RT112 and its chemotherapy­adapted sublines was investigated by MTT assay. The expression of anti­apoptotic proteins was determined by western blotting and the individual roles of cellular inhibitor of apoptosis protein (cIAP)1, cIAP2, x­linked inhibitor of apoptosis protein (XIAP) and induced myeloid leukemia cell differentiation protein (Mcl­1) were investigated by siRNA­mediated depletion. In particular, the bladder cancer sublines that were resistant to gemcitabine and cisplatin were cross­resistant to TRAIL. Resistant cells displayed upregulation of anti­apoptotic molecules compared with the parental cell line. Treatment with the second mitochondrial activator of caspases (SMAC) mimetic LCL­161 that antagonizes cIAP1, cIAP2 and XIAP resensitized chemoresistant cells to TRAIL. The resensitization of tumor cells to TRAIL was confirmed by depletion of antiapoptotic proteins with siRNA. Collectively, the findings of the present study demonstrated that SMAC mimetic LCL­161 increased the sensitivity of the parental cell line RT112 and chemotherapy­resistant sublines to TRAIL, suggesting that inhibiting anti­apoptotic molecules renders TRAIL therapy highly effective for chemotherapy­sensitive and ­resistant urothelial cancer cells.

Genomically informed small-molecule drugs overcome resistance to a sustained-release formulation of an engineered death receptor agonist in patient-derived tumor models.

Extrinsic pathway agonists have failed repeatedly in the clinic for three core reasons: Inefficient ligand-induced receptor multimerization, poor pharmacokinetic properties, and tumor intrinsic resistance. Here, we address these factors by (i) using a highly potent death receptor agonist (DRA), (ii) developing an injectable depot for sustained DRA delivery, and (iii) leveraging a CRISPR-Cas9 knockout screen in DRA-resistant colorectal cancer (CRC) cells to identify functional drivers of resistance. Pharmacological blockade of XIAP and BCL-XL by targeted small-molecule drugs strongly enhanced the antitumor activity of DRA in CRC cell lines. Recombinant fusion of the DRA to a thermally responsive elastin-like polypeptide (ELP) creates a gel-like depot upon subcutaneous injection that abolishes tumors in DRA-sensitive Colo205 mouse xenografts. Combination of ELPdepot-DRA with BCL-XL and/or XIAP inhibitors led to tumor growth inhibition and extended survival in DRA-resistant patient-derived xenografts. This strategy provides a precision medicine approach to overcome similar challenges with other protein-based cancer therapies.

Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy.

We have recently investigated the reactivity of aryl-fluorosulfates as warheads to form covalent adducts with Lys, Tyr, and His residues. However, the rate of reaction of aryl-fluorosulfates seemed relatively slow, putting into question their effectiveness to form covalent adducts in cell. Unlike the previously reported agents that targeted a relatively remote Lys residue with respect to the target's binding site, the current agents were designed to more directly juxtapose an aryl-fluorosulfate with a Lys residue that is located within the binding pocket of the BIR3 domain of X-linked inhibitor of apoptosis protein (XIAP). We found that such new agents can effectively and rapidly form a covalent adduct with XIAP-BIR3 in vitro and in cell, approaching the rate of reaction, cellular permeability, and stability that are similar to what attained by acrylamides when targeting Cys residues. Our studies further validate aryl-fluorosulfates as valuable Lys-targeting electrophiles, for the design of inhibitors of both enzymes and protein-protein interactions.

Dual Antagonist of cIAP/XIAP ASTX660 Sensitizes HPV- and HPV+ Head and Neck Cancers to TNFα, TRAIL, and Radiation Therapy.

PURPOSE: Human papillomavirus-negative (HPV-) head and neck squamous cell carcinomas (HNSCC) harbor frequent genomic amplification of Fas-associated death domain, with or without concurrent amplification of Baculovirus inhibitor of apoptosis repeat containing (BIRC2/3) genes encoding cellular inhibitor of apoptosis proteins 1/2 (cIAP1/2). Antagonists targeting cIAP1 have been reported to enhance sensitivity of HPV-, but not HPV+ tumors, to TNF family death ligands (TNF and TRAIL) and radiation.Experimental Design: We tested a novel dual cIAP/XIAP antagonist ASTX660 in HPV+ and HPV- cell lines in combination with death ligands TNFα and TRAIL, and in preclinical xenograft models with radiation, an inducer of death ligands. The dependence of activity on TNF was examined by antibody depletion. RESULTS: ASTX660 sensitized subsets of HPV- and HPV+ HNSCC cell lines to TNFα and TRAIL. These antitumor effects of ASTX660 are the result of both apoptosis and/or necroptosis among HPV- cells, and primarily by apoptosis (caspase 3 and caspase 8 cleavage) in HPV+ cells. ASTX660 enhanced restoration of protein expression and inhibitory activity of proapoptotic tumor suppressor TP53 in HPV+ HNSCC. Furthermore, ASTX660 combined with radiotherapy, an inducer of death ligands, significantly delayed growth of both HPV- and HPV+ human tumor xenografts, an effect attenuated by anti-TNFα pretreatment blockade. CONCLUSIONS: IAP1/XIAP antagonist, ASTX660, sensitizes HPV+ HNSCC to TNFα via a mechanism involving restoration of TP53. These findings serve to motivate further studies of dual cIAP/XIAP antagonists and future clinical trials combining these antagonists with radiotherapy to treat both HPV+ and HPV- HNSCC.

4-HNE Induces Apoptosis of Human Retinal Pigment Epithelial Cells by Modifying HSP70.

The role of heat shock protein 70 (HSP70) in apoptosis of human retinal pigment epithelial cells (ARPE-19) induced by 4-hydroxy-2-nonenal (4-HNE) was explored. Different concentrations of 4-HNE were used to stimulate ARPE-19 cells, and apoptosis was measured by flow cytometry. The expression of apoptotic-related proteins, HSP70, X-linked inhibitor-of-apoptosis (XIAP), Bcl-2, and Bax were quantified by Western blotting. HSP70 and XIAP overexpression plasmids, or their corresponding siRNAs were transfected into ARPE-19 cells using Lipofectamine™ 2000. Co-immunoprecipitation and Western blotting were used to detect the effect of 4-HNE on the expression of HSP70 and the binding level between 4-HNE and HSP70. The results showed that 4-HNE induced late apoptosis in ARPE-19 cells, accompanied by elevated levels of 4-HNE-modified HSP70, but it did not affect HSP70 protein expression. 4-HNE-modified HSP70 down-regulated the expression of the apoptosis inhibitory protein XIAP. Overexpression of HSP70 or XIAP inhibited 4-HNE-induced apoptosis of ARPE-19 cells. It was suggested that 4-HNE could promote XIAP degradation by modification of HSP70 to induce late apoptosis of human retinal pigment epithelial cells.

SM­164 enhances the antitumor activity of adriamycin in human U2­OS cells via downregulation of X­linked inhibitor of apoptosis protein.

The antitumor effects of SM­164 and adriamycin (ADM) on human osteosarcoma U2­OS cells, the underlying mechanism are yet to be investigated. In the present study, U2­OS cells were divided into control, ADM, SM­164, and ADM + SM­164 groups. In addition, cells treated with both SM­164 and ADM were further divided into three subgroups: SM­164 + ADM, SM­164 + ADM + vector and SM­164 + ADM + X­linked inhibitor of apoptosis protein (XIAP) silencing groups. XIAP expression was achieved via transfection with shRNA lentiviral vectors. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to detect the expression of caspases­7, ­9, and ­3, poly ADP­ribose polymerase (PARP), XIAP, cellular inhibitor of apoptosis protein­1 (cIAP­1) and survivin. Cell viability and apoptosis were evaluated using MTT and flow cytometry assays, respectively. Compared with the control group, cell viability decreased, while apoptosis was increased in the ADM and SM­164­treatment group. ADM and SM­164 treatment promoted the expression of caspases­7, ­9 and ­3, and PARP, but reduced the expression of XIAP, survivin and cIAP­1. Compared with ADM + SM­164 group, XIAP silencing with ADM + SM­164 treatment further reduced cell viability, promoted apoptosis, increased caspase­7, ­9 and ­3, and PARP expression; however the expression of survivin and cIAP­1 were reduced. Combined ADM and SM­164 treatment may be considered as potential therapeutic agent in the treatment of osteosarcoma, possibly via reductions XIAP expression.