XIAP Levels Research Articles

Evodiamine inhibits proliferation and induces apoptosis of nasopharyngeal carcinoma cells via the SRC/ERBB2-mediated MAPK/ERK signaling pathway

This study aimed to investigate the effect and potential mechanism of evodiamine (EVO) on proliferation and apoptosis of nasopharyngeal carcinoma (NPC) cells. EVO inhibited proliferation, blocked cell cycle progression, and induced apoptosis of NPC cells. There are 27 known anti-NPC targets of EVO, of which eight are core targets, namely SRC, ERBB2, STAT3, MAPK8, NOS3, CXCL8, APP, and HDAC1. Molecular docking analysis showed that the binding of EVO with its key targets (SRC, ERBB2) was good. EVO also reduced the expression of SRC and ERBB2, the key proteins p-MEK and p-ERK1/2 of the MAPK/ERK signaling pathway, and the downstream proteins PCNA and XIAP. EVO inhibited the growth of NPC xenografts in nude mice and reduced the expression levels of SRC, ERBB2, ERK1/2, p-ERK1/2, PCNA and XIAP in NPC tissue. When the MAPK/ERK signaling pathway was activated by epidermal growth factor (EGF), the expression levels of PCNA and XIAP increased, the cell proliferation index increased, and the apoptosis rate decreased in the EGF + EVO treatment group compared to treatment with EVO alone. These changes indicated that the inhibitory effect of EVO on proliferation and apoptosis of NPC cells was related to the down-regulation of SRC and ERBB2 expression, and further inhibition of the MAPK/ERK signaling pathway.

Mechanism of Preventing Recurrence of Stage II-III Colorectal Cancer Metastasis with Immuno-inflammatory and Hypoxic Microenvironment by a Four Ingredients Chinese Herbal Formula: A Bioinformatics and Network Pharmacology Analysis.

Colorectal Cancer (CRC) is one of the top three malignancies with the highest incidence and mortality. The study aimed to identify the effect of Traditional Chinese Medicine (TCM) on postoperative patients with stage II-III CRC and explore the core herb combination and its mechanism. An observational cohort study was conducted on patients diagnosed with stage II-III CRC from January 2016 to January 2021. The primary outcome was disease-free survival, which was compared between the patients who received TCM or not, and the secondary outcome was the hazard ratio. The relevance principle was used to obtain the candidate herb combinations, and the core combination was evaluated through an assessment of efficacy and representativeness. Then, biological processes and signaling pathways associated with CRC were obtained by Gene Ontology function, Kyoto Encyclopedia of Gene and Genomes pathway, and Wikipathway. Furthermore, hub genes were screened by the Kaplan-Meier estimator, and molecular docking was employed to predict the binding sites of key ingredients to hub genes. The correlation analysis was employed for the correlations between the hub genes and tumor-infiltrating immune cells and hypoxiarelated genes. Ultimately, a quantitative polymerase chain reaction was performed to verify the regulation of hub genes by their major ingredients. A total of 707 patients were included. TCM could decrease the metastatic recurrence associated with stage II-III CRC (HR: 0.61, log-rank P < 0.05). Among those patients in the TCM group, the core combination was Baizhu → Yinchen, Chenpi, and Fuling (C combination), and its antitumor mechanism was most likely related to the regulation of BCL2L1, XIAP, and TOP1 by its key ingredients, quercetin and tangeretin. The expression of these genes was significantly correlated with both tumor-infiltrating immune cells and hypoxia- related genes. In addition, quercetin and tangeretin down-regulated the mRNA levels of BCL2L1, XIAP, and TOP1, thereby inhibiting the growth of HCT116 cells. Overall, a combination of four herbs, Baizhu → Yinchen, Chenpi, and Fuling, could reduce metastatic recurrence in postoperative patients with stage II-III CRC. The mechanism may be related to the regulation of BCL2L1, XIAP, and TOP1 by its key ingredients quercetin and tangeretin.

Abstract 6203: An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients

Abstract Background: Drug resistance is a major challenge in inflammatory breast cancer (IBC), a hyperproliferative subtype characterized by the presence of diffuse tumor cell clusters that show high level of heterogeneity. As the degree of tumor heterogeneity during cancer progression can cause evolution of cell states that can either compete or co-operate resulting in therapeutic failure, the study objective was to identify drug resistance networks that can serve as biomarkers or therapeutic targets. Methods: Integrated transcriptomic and proteomic profiling approach was conducted in a progression model of acquired drug resistance and its reversal comprised of a treatment-naïve patient-derived SUM149 cell line, along with two isogenic derivatives and representing drug-resistant (rSUM149) and resistance-reversal states (rrSUM149). Global gene expression analysis in IBC patients (n=87) recorded responses to neoadjuvant chemotherapy. Based on the data, targeted multikinase inhibitors were evaluated in viability and signaling assays. Results: We identified specific ribosomal proteins associated with resistance acquisition, which correlated with high levels of pERK, CDK1, XIAP, and SOD2. Conversely, the resistance reversal in rrSUM149 showed almost complete normalization to the profile of SUM149. However, VIPER analysis revealed proteins related to ribosomal processes (AGO2, Exportin 1, RPL5) that did not revert in rrSUM149, suggesting this pathway may have pleiotropic effects in governing drug resistance. Notably, genes linked to ribosomal processes were significantly enriched (P&amp;lt;0.001) among the overexpressed genes in IBC patients (n=87) who exhibited pCR to neoadjuvant chemotherapy. This is crucial as IBC tumors, including rSUM149, exhibit MAPK hyperactivation including in the presence of EGFR tyrosine kinase inhibitors. Treatment with Merestinib, a multikinase inhibitor, suppressed pERK, peIF4E, downstream targets like CDK1, and ribosomal protein IMP3 underscoring the impact on protein synthesis signaling crucial for countering translational dependence in cancer cells. Additionally, Merestinib synergized with EGFR-TKI (lapatinib) in increasing cell death in all three states. Conclusion: Overall, our study describes adaptive changes in response to both therapeutic stress and therapy break that have the potential to serve as biomarkers or extended for pharmacological interrogation toward precision medicine for this rare, understudied cancer. Furthermore, the ability of Merestinib, in clinical trials, to target translational dependence is attractive as cancer cells often appropriate these signaling pathways to gain tolerance to various stress stimuli. Support: NIH-R01CA264529; DoD Breast Cancer Breakthrough level 2 Award W81XWH2010153 Citation Format: Gayathri R. Devi, Pritha PAI, Matthew W. Foster, Dorababu S. Sannareddy, Seayoung Lee, Steven V. Laere. An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6203.

Moracin D suppresses cell growth and induces apoptosis via targeting the XIAP/PARP1 axis in pancreatic cancer

BackgroundPancreatic cancer, a tumor with a high metastasis rate and poor prognosis, is among the deadliest human malignancies. Investigating effective drugs for their treatment is imperative. Moracin D, a natural benzofuran compound isolated from Morus alba L., shows anti-inflammation and anti-breast cancer properties and is effective against Alzheimer's disease. However, the effect and mechanism of Moracin D action in pancreatic cancer remain obscure. PurposeTo investigate the function and molecular mechanism of Moracin D action in repressing the malignant progression of pancreatic cancer. MethodsPancreatic cancer cells were treated with Moracin D, and cell proliferation was evaluated by cell counting kit-8 (CCK-8) and immunofluorescence assays. The clonogenicity of pancreatic cancer cells was assessed based on plate colony formation and soft agar assay. Flow cytometry was used to detect cell apoptosis. The expression of proteins related to the apoptosis pathway was determined by Western blot analysis. Moracin D and XIAP were subjected to docking by auto-dock molecular docking analysis. Ubiquitination levels of XIAP and the interaction of XIAP and PARP1 were assessed by co-immunoprecipitation analysis. Moracin D's effects on tumorigenicity were assessed by a tumor xenograft assay. ResultsMoracin D inhibited cell proliferation, induced cell apoptosis, and regulated the protein expression of molecules involved in caspase-dependent apoptosis pathways. Moracin D suppressed clonogenicity and tumorigenesis of pancreatic cancer cells. Mechanistically, XIAP could interact with PARP1 and stabilize PARP1 by controlling its ubiquitination levels. Moracin D diminished the stability of XIAP and decreased the expression of XIAP by promoting proteasome-dependent XIAP degradation, further blocking the XIAP/PARP1 axis and repressing the progression of pancreatic cancer. Moracin D could dramatically improve the chemosensitivity of gemcitabine in pancreatic cancer cells. ConclusionMoracin D repressed cell growth and tumorigenesis, induced cell apoptosis, and enhanced the chemosensitivity of gemcitabine through the XIAP/PARP1 axis in pancreatic cancer. Moracin D is a potential therapeutic agent or adjuvant for pancreatic cancer.

Enhanced translocation of TRIM32 to mitochondria sensitizes dopaminergic neuronal cells to apoptosis during stress conditions in Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease characterized by progressive loss of dopamine-producing neurons from the substantia nigra region of the brain. Mitochondrial dysfunction is one of the major causes of oxidative stress and neuronal cell death in PD. E3 ubiquitin ligases such as Parkin (PRKN) modulate mitochondrial quality control in PD; however, the role of other E3 ligases associated with mitochondria in the regulation of neuronal cell death in PD has not been explored. The current study investigated the role of TRIM32, RING E3 ligase, in sensitization to oxidative stress-induced neuronal apoptosis. The expression of TRIM32 sensitizes SH-SY5Y dopaminergic cells to rotenone and 6-OHDA-induced neuronal death, whereas the knockdown increased cell viability under PD stress conditions. The turnover of TRIM32 is enhanced under PD stress conditions and is mediated by autophagy. TRIM32 translocation to mitochondria is enhanced under PD stress conditions and localizes on the outer mitochondrial membrane. TRIM32 decreases complex-I assembly and activity as well as mitochondrial reactive oxygen species (ROS) and ATP levels under PD stress. Deletion of the RING domain of TRIM32 enhanced complex I activity and rescued ROS levels and neuronal viability under PD stress conditions. TRIM32 decreases the level of XIAP, and co-expression of XIAP with TRIM32 rescued the PD stress-induced cell death and mitochondrial ROS level. In conclusion, turnover of TRIM32 increases during stress conditions and translocation to mitochondria is enhanced, regulating mitochondrial functions and neuronal apoptosis by modulating the level of XIAP in PD.

Alpha-santalol, a derivative of sandalwood oil prevents development of prostate cancer in TRAMP mice

BackgroundAlpha-santalol, a major component of sandalwood oil inhibits growth of cultured prostate cancer cells in vitro by causing apoptosis. However, its in vivo efficacy against prostate cancer development is not established. Hypothesis/purposeProstate cancer is one of the most commonly diagnosed cancers in men. The present study was undertaken to determine the in vivo efficacy of alpha-santalol using TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice as a model. MethodsTRAMP mice were used for the in vivo studies. Immunohistochemical analysis was used to assess prostate/tumor sections and Ki-67 staining. Western blot analysis was used to determine expression of various proteins. TUNEL assay was used to detect apoptotic bodies. ResultsAdministration of alpha-santalol (100 mg/kg body weight) decreased the incidence of prostate tumors, average wet weights of urogenital organs and prostate weight compared to control mice. In addition, the area occupied by normal prostate was significantly higher, along with poorly differentiated carcinoma that was significantly lower in the dorsolateral prostate sections of alpha-santalol-treated mice compared with the controls. Furthermore, the dorsolateral sections of prostate from alpha-santalol-treated mice exhibited decreased cell proliferation (Ki-67 staining) in association with induction of apoptosis (TUNEL-positive cells). In agreement with immunohistochemical analysis, Western blotting analysis of prostate/tumor samples from alpha-santalol-treated group revealed a decrease in survivin, XIAP, PCNA, cyclin D and CDC2 levels compared to control samples. ConclusionThe present study shows for the first time, that alpha-santalol administration inhibits the development of prostate cancer in TRAMP mice by decreasing cell proliferation and inducing apoptosis, and warrants future studies for its clinical development.

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.

Microglia Regulating Neuronal Apoptosis Via Alpha7 Nicotinic Acetylcholine Receptor in HIV-Associated Neurocognitive Disorders

Background: Microglia, the innate immune cells of the central nervous system (CNS), originate from erythromyeloid progenitor cells in the embryonic yolk sac. As the innate immune cells of the CNS, microglia play an important role in innate immunity, neurogenesis, and brain tissue repair by activating the alpha7 nicotinic acetylcholine receptor (α7nAChR). They are the key target cells for HIV-1 infection in the CNS and have been shown to produce a chronic inflammatory response in the brain which underlies HIV-associated neurocognitive disorders (HAND). Apart from the microglial neuroinflammation, recent studies have reported that activated microglia could induce neuronal apoptosis through the release of neurotoxic factors (e.g., ROS and NLRP3 inflammasome) in HAND. In addition, α7nAChR was significantly up-regulated in the striatum which is the most serious part of neuron apoptosis in gp120 transgenic mice brains. However, the underlying mechanism of neuronal cell apoptosis by gp120-activated microglia remains unclear. Here, this study aimed to explore the mechanism by which HIV-1 gp120-activated microglia induce neuronal apoptosis. Materials and methods: The expression of α7nAChR was examined by immunohistochemistry in the brain of HIV-1 LAV gp120 transgenic (Tg) mice (C57BL/6J background, 12-month-old). Methyllycaconitine (MLA, specific α7nAChR agonists) and Nicotine (NT, specific α7nAChR antagonists) were used to evaluate the effect of α7nAChR on apoptosis induced by gp120. Immortalized microglial cells (BV2) were incubated for 24 hours with gp120, NT, and gp120+NT, respectively, with or without preincubation with MLA for 15 minutes to obtain their supernatants. Next, the neuronal cells (SH-SY5Y) were treated with the same as BV2 or with collected BV2 culture supernatants. The apoptosis effect of SH-SY5Y and the level of ROS in the BV2 culture supernatants were assessed via quantitative analysis of flow cytometry. Comparisons between supernatant groups and drug groups were conducted to evaluate the effects of the SH-SY5Y apoptosis induced by microglia. Western blotting of microglia cell lysates was employed for the analysis of cellular signaling pathways. Student's t-tests and ANOVA were applied to determine statistical significance. Results: The expression of α7nAChR in HIV-1 gp120 transgenic mice was significantly upregulated (P < 0.001). The effect of apoptosis was more significant in microglia culture supernatants groups compared to that in drug-treated groups (P < 0.001). Neuronal activity was not changed in the NT group or MLA group than in the control, but HIV-1 gp120 could increase the number of apoptotic neurons (P < 0.01). Significant neuronal apoptosis was observed when treated with BV2 supernatants from the group treated by combining the gp120 with NT (P < 0.001), and this effect was in turn inhibited by MLA (P < 0.001). These results suggested that gp120-activated microglia might induce neuronal apoptosis invitro owing to gp120 upregulating α7nAChR. In addition, the ROS level in microglia was significantly upregulated following gp120 exposure (P < 0.001), and this effect could be enhanced by NT (P < 0.001). Notably, there was a positive correlation between the levels of ROS in microglia culture supernatants and the levels of neuronal apoptosis treated in the same supernatants. This positive relevance indicated that microglia might induce neuronal apoptosis by producing excessive ROS. The expressions of p53, OMI, and caspase-3 significantly upregulated following gp120 exposure with the down-regulate expression of XIAP (P < 0.001). And this effect could be enhanced by NT (P < 0.001). The results of western blotting indicated that gp120 and NT can up-regulate the ROS to activate p53 by α7nAChR, which could lead to an increased OMI and decreased XIAP levels. (P < 0.001). Conclusions: Microglia may play a critical role in regulating neuronal apoptosis in HAND. HIV-1 gp120 and NT could increase the generation of ROS through α7nAChR in microglia, which leads to neuronal apoptosis. Moreover, our results showed that the combined action of NT and gp120 may upregulate p53 and Omi / HtrA2 protein expression, and the downregulation of XIAP protein may significantly activate the level of caspase-3. (Acknowledgements:Corresponding author: Hong Cao, gzhcao@smu.edu.cn; National Natural Science Foundation of China, No. 82172259 to H.C.) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

USP7 targets XIAP for cancer progression: Establishment of a p53-independent therapeutic avenue for glioma.

Ubiquitin specific peptidase 7 (USP7) is a deubiquitinating enzyme (DUB) that removes ubiquitin tags from specific target protein substrates in order to alter their degradation rate, sub-cellular localization, interaction, and activity. The induction of apoptosis upon USP7 inhibition is well established in cancer containing wild type p53, which operates through the 'USP7-Mdm2-p53' axis. However, in cancers without functional p53, USP7-dependent apoptosis is induced through many other alternative pathways. Here, we have identified another critical p53 independent path active under USP7 to regulate apoptosis. Proteomics analysis identifies XIAP as a potential target of USP7-dependent deubiquitination. GSEA analysis revealed up-regulation of apoptosis signalling upon USP7 inhibition associated with XIAP down-regulation. Modulation of USP7 expression and activity in multiple cancer cell lines showed that USP7 deubiquitinates XIAP to inhibit apoptosis in a caspase-dependent pathway, and the combinatorial inhibition of USP7 and XIAP induces apoptosis in vitro and in vivo. Immunohistochemical staining revealed that grade-wise accumulation of USP7 correlated with an elevated level of XIAP in glioma tissue. This is the first report on the identification and validation of XIAP as a novel substrate of USP7 and together, they involve in the empowerment of the tumorigenic potential of cancer cells by inhibiting apoptosis.

Endogenous TRAIL-R4 critically impacts apoptotic and non-apoptotic TRAIL-induced signaling in cancer cells.

Binding of TRAIL to its death domain-containing receptors TRAIL-R1 and TRAIL-R2 can induce cell death and/or pro-inflammatory signaling. The importance of TRAIL and TRAIL-R1/R2 in tumor immune surveillance and cancer biology has meanwhile been well documented. In addition, TRAIL has been shown to preferentially kill tumor cells, raising hope for the development of targeted anti-cancer therapies. Apart from death-inducing receptors, TRAIL also binds to TRAIL-R3 and TRAIL-R4. Whereas TRAIL-R3 is lacking an intracellular domain entirely, TRAIL-R4 contains a truncated death domain but still a signaling-competent intracellular part. It is assumed that these receptors have anti-apoptotic, yet still not well understood regulatory functions.To analyze the significance of the endogenous levels of TRAIL-R4 for TRAIL-induced signaling in cancer cells, we stably knocked down this receptor in Colo357 and MDA-MB-231 cells and analyzed the activation of apoptotic and non-apoptotic pathways in response to treatment with TRAIL.We found that TRAIL-R4 affects a plethora of signaling pathways, partly in an opposite way. While knockdown of TRAIL-R4 in Colo357 strongly increased apoptosis and reduced clonogenic survival, it inhibited cell death and improved clonogenic survival of MDA-MB-231 cells after TRAIL treatment. Furthermore, TRAIL-R4 turned out to be an important regulator of the expression of a variety of anti-apoptotic proteins in MDA-MB-231 cells since TRAIL-R4-KD reduced the cellular levels of FLIPs, XIAP and cIAP2 but upregulated the levels of Bcl-xL. By inhibiting Bcl-xL with Navitoclax, we could finally show that this protein mainly accounts for the acquired resistance of MDA-MB-231 TRAIL-R4-KD cells to TRAIL-induced apoptosis. Analyses of non-apoptotic signaling pathways revealed that in both cell lines TRAIL-R4-KD resulted in a constitutively increased activity of AKT and ERK, while it reduced AKT activity after TRAIL treatment. Furthermore, TRAIL-R4-KD potentiated TRAIL-induced activation of ERK and p38 in Colo357, and NF-κB in MDA-MB-231 cells. Importantly, in both cell lines the activity of AKT, ERK, p38 and NF-κB after TRAIL treatment was higher in TRAIL-R4-KD cells than in respective control cells.Thus, our data provide evidence for the important regulatory functions of endogenous TRAIL-R4 in cancer cells and improve our understanding of the very complex human TRAIL/TRAIL-R system.

Allyl Isothiocyanate (AITC) Induces Apoptotic Cell Death In Vitro and Exhibits Anti-Tumor Activity in a Human Glioblastoma GBM8401/luc2 Model.

Some clinically used anti-cancer drugs are obtained from natural products. Allyl isothiocyanate (AITC), a plant-derived compound abundant in cruciferous vegetables, has been shown to possess an anti-cancer ability in human cancer cell lines in vitro, including human brain glioma cells. However, the anti-cancer effects of AITC in human glioblastoma (GBM) cells in vivo have not yet been examined. In the present study, we used GBM8401/luc2 human glioblastoma cells and a GBM8401/luc2-cell-bearing animal model to identify the treatment efficacy of AITC. Here, we confirm that AITC reduced total cell viability and induced cell apoptosis in GBM8401/luc2 cells in vitro. Furthermore, Western blotting also showed that AITC induced apoptotic cell death through decreased the anti-apoptotic protein BCL-2, MCL-1 expression, increased the pro-apoptotic protein BAX expression, and promoted the activities of caspase-3, -8, and -9. Therefore, we further investigated the anti-tumor effects of AITC on human GBM8401/luc2 cell xenograft mice. The human glioblastoma GBM8401/luc2 cancer cells were subcutaneously injected into the right flank of BALB/c nude mice to generate glioblastoma xenograft mice. The animals were randomly divided into three groups: group I was treated without AITC (control); group II with 0.1 mg/day of AITC; and group III with 0.2 mg/day of AITC every 3 days for 27 days. Bodyweight, and tumor volume (size) were recorded every 3 days. Tumors exhibiting Luc2 intensity were measured, and we quantified intensity using Living Image software on days 0, 12, and 24. After treatment, tumor weight from each mouse was recorded. Tumor tissues were examined for histopathological changes using H&E staining, and we analyzed the protein levels via immunohistochemical analysis. Our results indicate that AITC significantly inhibited tumor growth at both doses of AITC due to the reduction in tumor size and weight. H&E histopathology analysis of heart, liver, spleen, and kidney samples revealed that AITC did not significantly induce toxicity. Body weight did not show significant changes in any experiment group. AITC significantly downregulated the protein expression levels of MCL-1, XIAP, MMP-9, and VEGF; however, it increased apoptosis-associated proteins, such as cleaved caspase-3, -8, and -9, in the tumor tissues compared with the control group. Based on these observations, AITC exhibits potent anti-cancer activity in the human glioblastoma cell xenograft model via inhibiting tumor cell proliferation and the induction of cell apoptosis. AITC may be a potential anti-GBM cancer drug that could be used in the future.

Inhibition of noncaspase proteases, calpain and proteasome, via ALLN and Bortezomib contributes to cell death through low degradation of pro-/anti-apoptotic proteins and apoptosis induction.

Dysfunction at any regulatory point along the apoptotic signaling pathway is closely related to many diseases including cancers. The apoptotic protein expression level is an important cause of cancer-related death, and the correct degradation of apoptotic proteins is involved in tumor development. Therefore, understanding of a regulatory point that underlying cancer-related death may help the development of new strategies to overcome the clinical challenges. Here, proteasome inhibitor Bortezomib and calpain inhibitor ALLN were examined on protein levels of caspase-3, caspase-9, XIAP, and E3-ligase PARC in HEK293T cells overexpressing XIAP and caspase-9. ATP depletion and caspase-3 activation were as a consequence of Bortezomib and ALLN function. Higher numbers of PI-stained cells provided evidence of cell death by both inhibitors. Western blotting analysis showed that both ALLN and Bortezomib equally inhibited degradation of XIAP, but only ALLN was effective at inhibiting caspase proteolytic degradation. Moreover, treatment of cells with both types of inhibitors significantly increased the level of E3-ligase PARC. Our findings showed that inhibition of proteasome and calpains enhanced the level of anti-apoptotic, XIAP and PARC, and pro-apoptotic, caspase-9 and 3 proteins, which totally promote cell death significantly.

Abstract 3008: Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition

Abstract Cell proliferation and cell death are essential for maintaining homeostatic balance in eukaryotic organisms. Many diseases develop when cellular homeostasis is disturbed. Currently, several anti-cancer drugs are known to induce apoptosis through activation of intrinsic or extrinsic pathways. However, many types of cancer cells have acquired resistance due to quick adaptation to resist pro-apoptotic mechanisms. Therefore, simultaneous induction of other forms of cell deaths such as necroptosis, autophagy, etc., in addition to apoptosis, is becoming an attractive strategy for controlling tumor growth and metastasis. For this purpose, we have explored the role of XIAP in caspase-independent mechanisms of cell death during HDAC (Histone Deacetylase) inhibition. Our experiments with HDAC inhibitor SAHA (Suberoylanilide hydroxamic acid) have revealed that the anti-apoptotic biomarkers, including survivin and XIAP, exhibit important regulatory functions for inducing necroptosis in cancer cells. Interestingly, many of the signal adaptors of apoptosis and necroptosis are known to show dual functions during drug-induced cancer cell death. For example, the formation of the survivin-XIAP complex prevents XIAP from undergoing proteasomal degradation and polyubiquitination, thereby stabilizing XIAP in cancer cells. When the survivin level is increased, it is also known to stop the inhibition of XIAP by Smac. In addition, since XIAP can inhibit caspase -9 and the executioner caspases -3 and -7, the apoptosis mechanisms will be blocked, and cell proliferation will continue to promote cell survival and tumorigenesis. Our western blot data revealed that the caspase -9, -3, -7, and -8 levels were significantly downregulated in MCF-7 breast cancer cells following SAHA treatment. Furthermore, SAHA treatment also increased the levels of necroptosis inducers such as phospho-RIP3 and MLKL in MCF-7 cells. However, treatment of the same MCF-7 cells with other drugs such as RG-7388, an MDM2 inhibitor, was able to induce apoptosis through activation of caspases. Our current results show that MCF-7 cells may switch to the necroptosis pathway due to the inhibition of caspases and increased levels of survivin and XIAP. (This research was supported by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Umamaheswari Natarajan, Grace Waldron, Thiagarajan Venkatesan, Appu Rathinavelu. Induction of necroptosis through caspase-independent mechanisms in MCF-7 breast cancer cells following HDAC inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3008.

LncRNA GIHCG promoted the proliferation and migration of renal cell carcinoma through regulating miR-499a-5p/XIAP axis

BackgroundOur previous study demonstrated that lncRNA GIHCG is upregulated in renal cell carcinoma (RCC) and that knockdown of lncRNA GIHCG suppresses the proliferation and migration of RCC cells. However, the mechanism of lncRNA GIHCG in RCC needs further exploration. MethodsThe proliferation, cell cycle, migration, and apoptosis of RCC cells were tested using CCK-8, flow cytometry, wound healing and Annexin-V/-FITC/PI flow cytometry assays, respectively. Dual-luciferase reporter and RNA pull-down or RNA immunoprecipitation assays (RIPs) were performed to analyze the interactions among lncRNA GIHCG, miR-499a-5p and XIAP. A tumour xenograft study was conducted to verify the function of lncRNA GIHCG in RCC development in vivo. ResultsKnockdown of lncRNA GIHCG inhibited cell proliferation and migration and induced G0/G1 arrest while promoting apoptosis. Overexpression of lncRNA GIHCG led to the opposite results. LncRNA GIHCG sponged miR-499a-5p and downregulated its expression in RCC cells. MiR-499a-5p overexpression suppressed RCC cell growth. MiR-499a-5p targeted XIAP and inhibited its expression. LncRNA GIHCG knockdown reduced the growth of tumour xenografts in vivo and the expression of XIAP while increasing miR-499a-5p levels. ConclusionLncRNA GIHCG accelerated the development of RCC by targeting miR-499a-5p and increasing XIAP levels.

A Dual Anti-Inflammatory and Anti-Proliferative 3-Styrylchromone Derivative Synergistically Enhances the Anti-Cancer Effects of DNA-Damaging Agents on Colon Cancer Cells by Targeting HMGB1-RAGE-ERK1/2 Signaling.

The current anti-cancer treatments are not enough to eradicate tumors, and therefore, new modalities and strategies are still needed. Most tumors generate an inflammatory tumor microenvironment (TME) and maintain the niche for their development. Because of the critical role of inflammation via high-mobility group box 1 (HMGB1)–receptor for advanced glycation end-products (RAGE) signaling pathway in the TME, a novel compound possessing both anti-cancer and anti-inflammatory activities by suppressing the HMGB1-RAGE axis provides an effective strategy for cancer treatment. A recent work of our group found that some anti-cancer 3-styrylchromones have weak anti-inflammatory activities via the suppression of this axis. In this direction, we searched such anti-cancer molecules possessing potent anti-inflammatory activities and discovered 7-methoxy-3-hydroxy-styrylchromone (C6) having dual suppressive activities. Mechanism-of-action studies revealed that C6 inhibited the increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) under the stimulation of HMGB1-RAGE signaling and thereby suppressed cytokine production in macrophage-like RAW264.7 cells. On the other hand, in colorectal cancer HCT116 cells, C6 inhibited the activation of ERK1/2, cyclin-dependent kinase 1, and AKT, down-regulated the protein level of XIAP, and up-regulated pro-apoptotic Bax and caspase-3/7 expression. These alterations are suggested to be involved in the C6-induced suppression of cell cycle/proliferation and initiation of apoptosis in the cancer cells. More importantly, in cancer cells, the treatment of C6 potentiates the anti-cancer effects of DNA-damaging agents. Thus, C6 may be a promising lead for the generation of a novel class of cancer therapeutics.

INHIBITOR OF APOPTOSIS XIAP AND GLIOBLASTOMA CELLS RESISTANCE TO VACCINIA VIRUS VV-GMCSF-LACT

It has been shown that under the action VV-GMCSF-Lact, the death of glioblastoma cells occurs mainly through the apoptotic pathway. When analyzing the complexes of Smac protein with the apoptosis inhibitor XIAP in human glioblastoma cells, it was found that in the cells most sensitive to VV-GMCSF-Lact, Smac level was increased after incubation with the virus. At the same time, overall level of XIAP was higher in more resistant cells.

NDUFC1 Is Upregulated in Gastric Cancer and Regulates Cell Proliferation, Apoptosis, Cycle and Migration.

Gastric cancer is one of the most common primary tumors of the digestive system. NADH: ubiquinone oxidoreductase subunit C1 (NDUFC1), which is an accessory subunit of the NADH dehydrogenase (complex I), is responsible for the transportation of electrons from NADH to the respiratory chain essential for the oxidative phosphorylation. However, little is known about the roles of NDUFC1 in carcinogenesis. In this study, NDUFC1 protein level in NSCLC tissues was tested by immunohistochemistry (IHC) staining. NDUFC1 mRNA level in gastric cancer cell lines was determined by qRT-PCR. MGC-803 and SGC-7901 cells were transfected with shNDUFC1 lentivirus designed to silence NDUFC1. MTT assay, CCK8 assay, wound healing assay and transwell migration assay were conducted. Cell cycle and apoptosis were detected by flow cytometry. In vivo experiments were performed using nude mice. The results indicated that overexpressed NDUFC1 in gastric cancer was related to more serious tumor infiltrates, a higher risk of lymphatic metastasis, a higher proportion of positive lymph nodes, and a more advanced tumor stage. Compared with shCtrl groups, MGC-803 and SGC-7901 of shNDUFC1 groups had lower abilities of proliferation and migration, higher levels of apoptosis. NDUFC1 knockdown also inhibited SGC-7901 cell growth in vivo and suppressed Ki67 expression in xenograft tumors. More importantly, we found that NDUFC1 downregulation made the levels of P-Akt, P-mTOR, CCND1, CDK6, PIK3CA, Bcl-2, Survivin, and XIAP decreased, and that PI3K/AKT signaling pathway agonist SC79 rescued the inhibitory effects on cell proliferation and migration, reversed the promoted effects on cell apoptosis caused by NDUFC1 knockdown. More importantly, compared with NDUFC1 knockdown group, the expression of P-Akt, Bcl-2, Survivin, and XIAP was raised in shNDUFC1 + SC79 group. Thus, our suspicion was that NDUFC1 exacerbates NSCLC progression via PI3K/Akt pathway. Taken together, our study indicated that targeting NDUFC1 could open innovative perspectives for new multi-targeting approaches in the treatment of gastric cancer.

Anti-Obesity Drug Orlistat Alleviates Western-Diet-Driven Colitis-Associated Colon Cancer via Inhibition of STAT3 and NF-κB-Mediated Signaling.

Many researchers have argued that Western diet (WD)-induced obesity accelerates inflammation and that inflammation is a link between obesity and colorectal cancer (CRC). This study investigated the effect of WDs on the development and progression of colitis-associated colon cancer (CAC) and the efficacy of the anti-obesity agent orlistat on WD-driven CAC in mice. The results revealed that the WD exacerbated CAC in azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced mice, which showed increased mortality, tumor formation, and aggravation of tumor progression. Furthermore, WD feeding also upregulated inflammation, hyperplasia, and tumorigenicity levels through the activation of STAT3 and NF-κB signaling in an AOM/DSS-induced mouse model. In contrast, treatment with orlistat increased the survival rate and alleviated the symptoms of CAC, including a recovery in colon length and tumor production decreases in WD-driven AOM/DSS-induced mice. Additionally, orlistat inhibited the extent of inflammation, hyperplasia, and tumor progression via the inhibition of STAT3 and NF-κB activation. Treatment with orlistat also suppressed the β-catenin, slug, XIAP, Cdk4, cyclin D, and Bcl-2 protein levels in WD-driven AOM/DSS-induced mice. The results of this study indicate that orlistat alleviates colon cancer promotion in WD-driven CAC mice by suppressing inflammation, especially by inhibiting STAT3 and NF-κB activation.

The SMAC mimetic AT-101 exhibits anti-tumor and anti-metastasis activity in lung adenocarcinoma cells by the IAPs/ caspase-dependent apoptosis and p65-NFƙB cross-talk.

Objective(s):The Inhibitors of Apoptosis (IAPs) regulate initiator and effector phases of caspase mediated apoptosis. This study evaluates the effects of SMAC mimetic AT-101 in regulation of IAPs/caspases/NFƙB-p65 in an adenocarcinoma cell line.Materials and Methods:MTT assay was performed in the NCI-H522 cell line. Flow cytometry was used for detecting cell cycle, apoptosis, and NFƙB-p65 regulation. Effects of AT-101 on IAPs and caspases were determined by quantitative real time-PCR and western blotting. AutoDock-VINA was used for computational analysis. Results:AT-101 reduced the cell proliferation of NCI-H522 with a GI50 value of 7 μM. The compound arrested adenocarcinoma cells in the G1 phase of the cell cycle and increased early and late phase apoptosis while decreasing tumor-cell trans-migration. AT-101 treatment to NCI H522 at a concentration of 0.35 μM decreased XIAP, cIAP-1, and cIAP-2 mRNA levels to 4.39±0.66, 1.93±0.26, and 2.20±0.24 folds, respectively. Increased dose of AT-101 at 0.7 μM concentration further decreased XIAP, cIAP-1, and cIAP-2 mRNA levels to 2.44±0.67, 1.46±0.93, and 0.97±0.10 folds, respectively. Similar effects of a dose-dependent decrease in the protein expressions of XIAP, cIAP-1, and cIAP-2 were observed with AT-101 treatments, while a dose-responsive increase in the mRNA and protein expression levels of caspase 6 and caspase 7 was observed in the NCI-H522 cell line. The compound exhibited binding affinity (-6.1 kcal/mol) and inhibited NFƙB-p65 in these cells. Conclusion:AT-101 had anti-tumor efficacy against lung adenocarcinoma cells which could be mediated through IAPs/caspase-dependent apoptosis and NFƙB-p65 cross talk. Results from this study suggests a signal cross talk between IAPs and NFkB and open new channels for further investigations in therapeutic intervention against lung cancer management.

Abstract 2583: Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo

Abstract Head and neck cancer (HNC) is the seventh most prevalent cancer worldwide. The five-year survival rate is less than fifty percent despite significant advances in therapeutic approaches. Early diagnosis and treatment can help in prevention and disease management. Studies have reported that dietary intake of Allium vegetables or processed garlic that contains diallyl trisulfide (DATS) lowered the risk of various cancers. However, it has not been investigated for its efficacy and molecular mechanisms against HNC. Herein, we investigated the effect of DATS on HNC UMSCC-22A, UMSCC-22B, and Cal33 cells in vitro. DATS treatment significantly reduced the cell viability of HNC cells. DATS induced significant G2/M phase cell cycle arrest, which resulted in the accumulation of cyclin B1 and induced levels of p21. DATS-induced M phase arrest was attenuated by N-acetyl cysteine (NAC) treatment, which suggested that DATS mediates growth-suppressive effects by reactive oxygen species (ROS) generation. DATS treatment induced mitochondrial dysfunction. DATS induced ROS production led to apoptosis, which was attenuated by NAC treatment. DATS increased proapoptotic protein cleaved caspase-3, cleaved PARP, and Bax/Bcl2 ratio and decreased the levels of antiapoptotic protein XIAP. DATS induced ROS mediated DNA damage which was attenuated by NAC treatment. Further, DATS mediated increase in level of cleaved PARP, accumulation of Cyclin B1 and decrease in XIAP was attenuated by NAC treatment. DATS (1-10 μM) treatment resulted in inhibition of self renewal of HNC cancer stem cells (HNC CSCs) sphere formation. DATS reduced aldehyde dehydrogenase 1 (ALDH1) activity and CD133high/CD44high HNC CSC fraction. DATS-treated SCID mice tumor xenograft also revealed it's in vivo efficacy on HNC CSCs. Further, DATS treatment reduced the tumor weight, volume and reduced the levels of Ki67 cell proliferation marker in UMSCC22B head and neck cancer CD1 nude mice tumor xenograft. DATS treatment decreased tumor incidence and inhibited tumor promotion and progression in twenty two week C57BL6 4-Nitroquinoline 1-oxide (4-NQO)-induced mouse oral carcinogenesis model. DATS treatment reduced the cell proliferation marker Ki67 and increased TUNEL positive apoptotic cells in treatment groups compared to control group. Collectively, DATS inhibited cell proliferation, induced cell cycle arrest, and cell death via apoptosis involving DNA damage, mitochondrial dysfunction, and ROS generation. DATS treatment also reduced the HNC CSC fraction, sphere formation and ALDH1 activity. More importantly, DATS inhibited cancer incidence and progression in 4-NQO oral carcinogenesis study. Further, DATS inhibited HNC tumor growth and HNC CSC fraction in vivo. Thus, DATS could be a potential chemopreventive and chemotherapeutic agent against head and neck cancer. Citation Format: Sivapar V. Mathan, Su-Hyeong Kim, Shivendra V. Singh, Rana P. Singh. Effects of garlic compound diallyl trisulfide on head and neck cancer cells and cancer stem cells in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2583.