Activation Of Apoptosis Pathway Research Articles

Immune cells and their inflammatory mediators modify β cells and cause checkpoint inhibitor-induced diabetes.

Checkpoint inhibitors (CPIs) targeting programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen 4 (CTLA-4) have revolutionized cancer treatment but can trigger autoimmune complications, including CPI-induced diabetes mellitus (CPI-DM), which occurs preferentially with PD-1 blockade. We found evidence of pancreatic inflammation in patients with CPI-DM with shrinkage of pancreases, increased pancreatic enzymes, and in a case from a patient who died with CPI-DM, peri-islet lymphocytic infiltration. In the NOD mouse model, anti–PD-L1 but not anti–CTLA-4 induced diabetes rapidly. RNA sequencing revealed that cytolytic IFN-γ+CD8+ T cells infiltrated islets with anti–PD-L1. Changes in β cells were predominantly driven by IFN-γ and TNF-α and included induction of a potentially novel β cell population with transcriptional changes suggesting dedifferentiation. IFN-γ increased checkpoint ligand expression and activated apoptosis pathways in human β cells in vitro. Treatment with anti–IFN-γ and anti–TNF-α prevented CPI-DM in anti–PD-L1–treated NOD mice. CPIs targeting the PD-1/PD-L1 pathway resulted in transcriptional changes in β cells and immune infiltrates that may lead to the development of diabetes. Inhibition of inflammatory cytokines can prevent CPI-DM, suggesting a strategy for clinical application to prevent this complication.

Pattern Recognition Receptors of Nucleic Acids Can Cause Sublethal Activation of the Mitochondrial Apoptosis Pathway during Viral Infection.

The mitochondrial apoptosis pathway has the function to kill the cell, but recent work shows that this pathway can also be activated to a sublethal level, where signal transduction can be observed but the cell survives. Intriguingly, this signaling has been shown to contribute to inflammatory activity of epithelial cells upon infection with numerous agents. This suggests that microbial recognition can generate sublethal activity in the mitochondrial apoptosis pathway. Because this recognition is achieved by pattern recognition receptors (PRRs), it also implies that PRR signals are linked to the mitochondrial apoptosis apparatus. We here test this hypothesis during infection of epithelial cells with modified vaccinia virus Ankara (MVA). MVA recognition is achieved through receptors specific for nucleic acids, and we present evidence that the three receptors, Toll-like receptor 3 (TLR3), RIG-I/MDA5, and cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING), are involved in this signaling. When stimulated directly by specific ligands, all three receptors could trigger sublethal apoptosis signals. During infection with MVA, sublethal apoptosis signals were unmasked in X-linked IAP (XIAP)-deficient cells, where apoptosis induction was observed. Deletion of any of the three signaling adapters, TRIF, MAVS, and STING, reduced the DNA damage response, a sensitive measure of sublethal apoptosis signals. Our results suggest that PRRs signal via mitochondria, where they generate sublethal signals through the BCL-2-family, which may contribute to the response to infectious agents. IMPORTANCE A contribution of the mitochondrial apoptosis apparatus, in the absence of cell death, to the reaction of nonprofessional immune cells to viruses is suggested to play a role as a broad alert system of an infected cell: the apoptosis system can be activated by many upstream signals and could therefore act as a central coordinator of viral recognition. The proapoptotic activity of PRRs has been documented in multiple situations, but this activity seems too low to be meaningful, and a physiological significance of such activity is not immediately obvious. This work suggests the alternative interpretation that PRRs do not have the primary function to induce apoptosis but to trigger sublethal signals in the apoptosis system. A number of lines of recent research suggest that mitochondria contribute to cellular reactions, and this pathway may be a way of triggering an early host response.

Hepatocellular cancer cell lines, Hep-3B and Hep-G2 display the pleiotropic response to resveratrol and berberine

PurposeHuman carcinoma cells with different p53 status exposed to a combination of bioactive substances, resveratrol and berberine, revealed different responses in cell viability via p53-dependant apoptosis pathway activation. Materials and methodsUsing 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, we investigated various and opposing effects in hepatocellular carcinoma cells, Hep-G2 and Hep-3B with different p53-status. ResultsCells decreased in viability after treatment with dose-dependent concentrations of resveratrol and berberine. Hep-3B p53 mutants were more sensitive in comparison to the p53 wild type Hep-G2 cell line. A synergistic effect was observed after treatment of Hep-3B cells with a combination of resveratrol/berberine ratios in favor of resveratrol (2:1, 3:1). The results suggest that an effective concentration of berberine, in the presence of resveratrol, could be decreased even to 50% (half the IC50 for berberine) in cancer treatment. Combined treatment with berberine and resveratrol, at the investigated concentrations and fractions, significantly reduces the viability of wild type p53 Hep-G2 and null p53-mutant Hep-3B cells by 20% and 40%, respectively. ConclusionsStronger toxic effects on viability and proliferation were observed in Hep-3B cells what is consistent with the assumptions that null p53-mutants activate apoptosis canonical pathway. In conclusion, p53 status in human hepatocellular cancer cell lines modulates responses to plant-derived therapies.

Evaluation of anticancer activity of ferrocene based benzothiazole and β-ketooxothioacetal

Ferrocene-based compounds have recently gained widespread importance in cancer treatment becuase of their lower toxicity and cost-effective synthesis. However, ferrocene-based compounds do not exist in nature. Moreover, the synthesis of thianes containing ferrocene is quite rare. In the recent past, our group has reported iodine catalyzed synthesis of ferrocenated benzothiazole and β-ketooxothioacetal by the thermal reaction of ferrocenated β-chlorocinnamaldehyde with 2-aminothiophenol and 2-mercaptoethanol respectively.The molecular structure of both the compounds is quite interesting due to the presence of ferrocene which makes them highly important from an anticancer point of view. In this script, the synergistic effect of ferrocene and thiol has been observed in ferrocene functionalized thiols (Fenton reagents) for tumour growth suppression. The anticancer properties of both the compounds separately as well as in combination show a significant anticancer effect on cancer cell lines HCT116 (Colon), A549 (lung cancer), and MCF7 (Breast cancer). Both compounds and their combinations reflect cell death by generating ROS and depolarization of mitochondrial membrane potential. Gene expression profiling and protein expression studies of apoptosis-related genes (cleaved caspase 9, cleaved caspase 3, cleaved PARP and Bcl-2) were also evaluated. Results indicated that ferrocenated benzothiozole and β-ketooxothioacetal induced apoptosis through oxidative stress-mediated activation of intrinsic apoptosis pathway by generating intracellular ROS, loss of MMP, and alteration of expression of genes related to apoptosis. In addition, the anti-metastatic activity of benzothiozole and β-ketooxothioacetal was seen by wound healing assay showing anti-migratory properties. The properties like anti metastatic activity, cytotoxicity and showing synergistic effect when used in combination make benzothiozole and β-ketooxothioacetal potent platforms for the development of new anticancer agents. Benzothiazole and β-ketooxothioacetal can replace existing organometallic compounds with rare metals like platinum, due to their higher stability, lower cost, and lower toxicity.

The molecular mechanisms of vulpinic acid induced programmed cell death in melanoma.

Malignant melanoma is an aggressive skin tumor with a rapidly increasing incidence and there is not yet a successful treatment strategy. Vulpinic acid (VA) is derived from secondary metabolites from lichen species. In the current study, we, for the first time, investigated the anti-cancer effects of VA and the underlying mechanism VA induced programmed cell death in melanoma. The anti-cancer effects of VA on melanoma cells were evaluated by the xCELLigence system, flow cytometry, caspase-3 activity and RT-PCR analysis. Our results showed that VA had a strong anti-proliferative effect on A-375 melanoma cells without damaging human epidermal melanocyte cells. Additionally, VA promoted apoptotic cell death through G2/M arrest and the activation of both intrinsic and extrinsic apoptosis pathways according to the analysis of 88 genes associated with apoptosis by qRT-PCR. Our findings suggest that VA could become an alternative topical and transdermal treatment strategy in the treatment of maligned melanoma cancer. However, further investigations are needed to assess the underlying molecular mechanism of VA mediated apoptotic cell death in the treatment of melanoma.

M1 macrophage-derived exosomes synergistically enhance the anti- bladder cancer effect of gemcitabine.

Gemcitabine (GEM) is one of the first choice drugs for treating bladder cancer. In this study, we loaded M1 macrophage-derived exosomes (M1-Exo) with GEM by ultrasonication technique to derive an M1-Exo-GEM drug delivery system, and then explored its effects on bladder cancer.After inducing M1 polarization of macrophages in vitro, ultracentrifugation was performed to obtain M1-Exo, followed by construction of M1-Exo-GEM via ultrasonication technique. Mouse bladder cancer MB49 cells were chosen for study. CCK-8, PI staining and flow cytometry (FCM) assays were employed to assess the cell viability and apoptosis level. Inflammatory cytokines were detected by ELISA, while the protein expressions of Bcl-2, Bax and Caspase-3 were examined through Western-Blotting. After injecting M1-Exo-GEM into the tumor-bearing mouse model, the pathological changes were observed by H&E staining, the cancer cell damage was detected by TUNEL staining, and the apoptosis pathway activation was analyzed through immunohistochemical (IHC) staining and protein expression assays for Caspase-3 and Bax.Our results showed that M1-Exo and GEM had cytotoxic effects on MB49 cells, which increased the apoptosis level and the inflammatory cytokine expressions. Compared to M1-Exo and GEM, M1-Exo-GEM was significantly more cytotoxic to MB49 cells while markedly up-regulating the expressions of inflammatory cytokines. In the tumor-bearing mouse model, M1-Exo-GEM significantly inhibited tumor growth and damaged tumor cells, which outperformed GEM. Meanwhile, it also increased the tissue levels of inflammatory cytokines.This study finds that the drug delivery system composed of M1-Exo and GEM can act synergistically with GEM to exert cytotoxicity and induce inflammatory damage of bladder cancer cells.

Protective Effect of Curcuma Extract in an Ex Vivo Model of Retinal Degeneration via Antioxidant Activity and Targeting the SUMOylation.

Retinal degeneration is the major and principal cause behind many incurable blindness diseases. Several studies indicated the neuroprotective effect of Curcuma longa in eye pathologies, specifically retinopathy. However, the molecular mechanism behind its effect has not been completely elucidated. Using an ex vivo model of retinal degeneration obtained from an ex vivo optic nerve cut (ONC), we demonstrated that Curcuma extract (Cur) exerted a neuroprotective effect. Importantly, Cur was able to modulate apoptosis and MAPK signaling pathway activation and prevent retinal ganglion cell (RGC) loss. Other well-known neuroprotective pharmacological tools, including memantine (Mem), citicoline (Cit), and ginkgolic acid (GA), were used to compare the potential mechanisms of Cur. The antioxidant activity of retinas treated with Cur following optic nerve cut was significantly higher than control, but Cur failed to change the retina glutamate content. Considering the antioxidant effect of Cur and taking advantage of our recent findings on the crosstalk between oxidative stress and post-translational protein modifiers, in particular, small ubiquitin-related modifier (SUMO), we were interested in exploring the effect of Cur on SUMOylation. We found that Cur significantly prevented the increase of protein SUMOylation, confirming our previous in vitro data indicating the cytoprotective effect of curcumin through modulating the oxidative stress and SUMO-JNK axis. Altogether, these results suggest that Curcuma protects the retina from degeneration via antioxidant activity and targets SUMOylation. Therefore, it might be considered for the combination therapy with other neuroprotective agents with different mechanisms in preclinical studies on retinal degeneration.

PS-NPs Induced Neurotoxic Effects in SHSY-5Y Cells via Autophagy Activation and Mitochondrial Dysfunction.

Polystyrene nanoparticles (PS-NPs) are organic pollutants that are widely detected in the environment and organisms, posing potential threats to both ecosystems and human health. PS-NPs have been proven to penetrate the blood–brain barrier and increase the incidence of neurodegenerative diseases. However, information relating to the pathogenic molecular mechanism is still unclear. This study investigated the neurotoxicity and regulatory mechanisms of PS-NPs in human neuroblastoma SHSY-5Y cells. The results show that PS-NPs caused obvious mitochondrial damages, as evidenced by inhibited cell proliferation, increased lactate dehydrogenase release, stimulated oxidative stress responses, elevated Ca2+ level and apoptosis, and reduced mitochondrial membrane potential and adenosine triphosphate levels. The increased release of cytochrome c and the overexpression of apoptosis-related proteins apoptotic protease activating factor-1 (Apaf-1), cysteinyl aspartate specific proteinase-3 (caspase-3), and caspase-9 indicate the activation of the mitochondrial apoptosis pathway. In addition, the upregulation of autophagy markers light chain 3-II (LC3-II), Beclin-1, and autophagy-related protein (Atg) 5/12/16L suggests that PS-NPs could promote autophagy in SHSY-5Y cells. The RNA interference of Beclin-1 confirms the regulatory role of autophagy in PS-NP-induced neurotoxicity. The administration of antioxidant N-acetylcysteine (NAC) significantly attenuated the cytotoxicity and autophagy activation induced by PS-NP exposure. Generally, PS-NPs could induce neurotoxicity in SHSY-5Y cells via autophagy activation and mitochondria dysfunction, which was modulated by mitochondrial oxidative stress. Mitochondrial damages caused by oxidative stress could potentially be involved in the pathological mechanisms for PS-NP-induced neurodegenerative diseases.

Duck Tembusu virus infection induces mitochondrial-mediated and death receptor-mediated apoptosis in duck embryo fibroblasts

Duck Tembusu virus (DTMUV) is a pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. Our previous study showed that DTMUV could induce duck embryo fibroblast (DEF) apoptosis, but the specific mechanism was not clear. In this study, we confirmed that DTMUV could induce the apoptosis of DEFs by DAPI staining and TUNEL staining. Furthermore, we found that the expression levels of cleaved-caspase-3/7/8/9 were significantly upregulated after DTMUV infection. After treatment of cells with an inhibitor of caspase-8 or caspase-9, DTMUV-induced apoptosis rates were significantly decreased, indicating that the caspase-8-mediated death receptor apoptotic pathway and caspase-9-mediated mitochondrial apoptotic pathway were involved in DTMUV-induced apoptosis. Moreover, we found that DTMUV infection not only caused the release of mitochondrial cytochrome C (Cyt C) and the downregulation of the apoptosis-inhibiting protein Bcl-2 but also reduced the mitochondrial membrane potential (MMP) and the accumulation of intracellular reactive oxygen species (ROS). Key genes in the mitochondrial apoptotic pathway and death receptor apoptotic pathway were upregulated to varying degrees, indicating the activation of the mitochondrial apoptosis pathway and death receptor apoptosis pathway. In conclusion, this study clarifies the molecular mechanism of DTMUV-induced apoptosis and provides a theoretical basis for revealing the pathogenic mechanism of DTMUV infection.

Ganoderma lucidum polysaccharide inhibits the proliferation of leukemic cells through apoptosis.

To investigate the cytotoxic effect of polysaccharides derived from Ganoderma lucidum on T lymphocyte leukemia cells. Water-soluble polysaccharides were extracted from the fruit bodies of G. lucidum, purified, and characterized using HPGPC-MALLS and NMR. The cytotoxicity of G. lucidum polysaccharide fraction 5 (GLP5) to T lymphocyte leukemia cell line Jurkatand human immortalized epidermal cell line HaCat was assessed using MTT assay. Apoptosis was assessed using flow cytometry. Expressions of apoptosis-related genes in the cells after being exposed to GLP5 were detected using Western blot assay. GLP5 was a β-(1→3) and β-(1→6) linked glucan. It inhibited the proliferation of Jurkat cells in a concentration-dependent manner and the half-maximal inhibitory concentration (IC50) was 34.5 mg/L but did not suppress the growth of HaCat cells. Apoptotic cells in Jurkatcells were detected to increase with increasing GLP5 concentrations. The expression levels of cleaved caspase-3 were significantly higher after the cells were exposed to 25 and 50 mg/L GLP5 when compared to non-exposed cells (Control). In addition, the expression levels of BAX and Bcl2 were significantly up- and down-regulated after treatment with GLP5 at 25 and 50 mg/L when compared with control (P<0.05), respectively. GLP5 has antiproliferative activity against Jurkat cells and the activity is likely mediated through the activation of apoptosis pathways.

Gemcitabine-Loaded Nanocarrier of Essential Oil from Pulicaria crispa: Preparation, Optimization, and In Vitro Evaluation of Anticancer Activity.

The limitations of gemcitabine (GEM) in cancer therapy are due to its poor pharmacokinetics, which cause undesired adverse effects. The current study was aimed at investigating the anticancer effect and apoptotic mechanism of synthesized nanoemulsion (NE) containing Pulicaria crispa essential oil (PC-EO) and GEM (PC-NE:GEM) on MCF-7 and Hep-G2 cancer cell lines. An optimized NE formulation was selected based on the Box–Behnken method. The droplet size of the optimized PC-NE was 9.93 ± 0.53 nm, but after GEM loading, it was increased to 11.36 ± 0.0.21 nm. Results from FTIR revealed that GEM was successfully loaded onto PC-NE. The antineoplastic effect of PC-NE:GEM on MCF-7 and Hep-G2 cancer cells was increased more than 100-fold relative to that of GEM. A combination index and isobologram based on CompuSyn software revealed the synergistic effect of the formulation produced by a 1:1 ratio combination of PC-NE and GEM. These findings were confirmed by examination of cellular morphologies. The combination formulation strongly induced about 4.48-fold and 2.95-fold increases in apoptosis in MCF-7 and Hep-G2 cells, respectively, when compared with GEM. Moreover, PC-NE:GEM produced a synergistic increase in ROS production in MCF-7 cells (15.23%) and Hep-G2 cells (31.69%), when compared with GEM. In addition, PC-NE:GEM enhanced the activation of the intrinsic apoptosis pathway through upregulation of expressions of p53 and Caspase-3, and downregulation of Bcl-2 expression in MCF-7 cells, while the expressions of Caspase-3, Bax, and p53 were upregulated in HepG2 cells. These results indicate that the GEM-loaded NE containing PC-EO may reduce the dose of GEM and eliminate the associated side effects.

Abstract 6180: First-in-human (FIH) phase I studies of SCB-313, a novel TNF-related apoptosis-inducing ligand TRAIL-Trimer™ fusion protein, for treatment of patients (pts) with malignant ascites (MA)

Abstract Background: SCB-313 is a recombinant human TRAIL-Trimer™ fusion protein engineered using a stabilized trimeric form of the TRAIL-protein. Binding of SCB-313 to the death receptors 4 and 5 leads to the activation of the extrinsic apoptosis pathway. Method: We performed a pooled analysis of two phase 1 studies conducted in Australia and China (NCT03443674 and NCT04051112, respectively). SCB-313 was given by intraperitoneal infusion 4 times at day D1, D4, D8, D11 in Australia study and D1, D8, D9, D10 in China study. Five dose cohorts: 10 mg, 20 mg, 40 mg, 80 mg, and 160 mg were evaluated using a sequential dose escalation design (accelerated titration dosing combined with 3+3 design) in pts with MA. The primary objective for both studies was safety and tolerability. Secondary objectives included PK/PD and efficacy. Results: 7 pts were enrolled in Australia (by data cutoff Mar 5th, 2021) and 12 pts in China (by data cutoff May 21st, 2021). 16 pts completed DLT observation period. Median age 57.6y, ECOG 1-2, 36.8% male. Colon cancer, n=5; ovarian cancer, n=4; breast cancer, n=3; gastric cancer, n=2; other cancers, n=5. Treatment-emergent adverse events (TEAE) were reported in 19 pts (100.0%). Most AEs were mild to moderate in severity (Grade 1 or 2). There were 9 (47.4%) pts who experienced Grade 3 AEs and no Grade 4 or 5 AEs were reported. No SCB-313 related SAEs or AEs leading to study discontinuations or death were reported. No DLT occurred. The most common TEAE included abdominal pain, pyrexia, abdominal distension. In Australia study, median (min, max) ascites flow rate of pts with MA (n=7) was 800 (400, 1091) ml/24h at baseline and 163.8 (-767, 412), 137.7 (-796.6, 392.6), -91.5 (-844.8, 517.5) and 120 (-47.92, 1105) ml/24h post first, second, third and the fourth SCB-313 administration respectively. In China study, the median flow rate at baseline (n=11) and post each subsequent SCB-313 dosing were respectively 723.1 (108.2, 1175), 12.1 (-335.1, 965.9), 84.2 (-442.5, 882), -13.9 (-404.4, 431.8) and -57.85 (-446.4, 2028) ml/24h. Estimated half-life of SCB-313 in peritoneal fluid after 1st injection is 3-4 hours (min, max: 1.85, 5.05). Cmax and AUC showed a nonlinear dose-dependent increase and near complete elimination from the peritoneal fluid by 24 hrs. In Australia study, apoptosis specific serum CK-18 concentrations increased following the first dose and remained above baseline for 5/7 pts. Conclusion: Although the MTD was not defined by these studies, SCB-313 therapy presents an acceptable safety profile at all tested dose levels. Measurable ascites flow rate decrease were observed at all dose- levels. These data support further development of SCB-313 for pts with MA. Citation Format: Ye Guo, Aflah Roohullah, Junli Xue, Wei Zhao, Morteza Aghmesheh, David Martin, Yu Zhou, Chao Gao, Yixuan Yang, Derek-Zhen Xu, Jin Li. First-in-human (FIH) phase I studies of SCB-313, a novel TNF-related apoptosis-inducing ligand TRAIL-Trimer™ fusion protein, for treatment of patients (pts) with malignant ascites (MA) [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 6180.

Abstract 1048: Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach

Abstract Background: Ewing's Sarcoma (ES) is a rare and aggressive pediatric bone tumor that is driven by a chromosomal translocation and fusion between EWS and FLI1 genes resulting in a chimeric protein. Nearly 85% of ES cases express the EWS/FLI protein which functions as a transcription factor and drives oncogenesis. EWS/FLI interacts with lysine-specific demethylase 1 (LSD1) that is highly expressed in pediatric sarcomas, to repress critical tumor suppressors. We have developed a potent reversible LSD1 inhibitor, SP-2577 (Seclidemstat), that shows the ability to impair tissue culture cell viability in multiple ES cell lines and is currently in clinical trials. A more favorable therapeutic response to SP-2577 may be obtained by combining drugs that target multiple pathways and/or inhibit resistance mechanisms. Experimental Design: In this study, we applied an arrayed CRISPR screening (Synthego) to identify survival genes that can be candidates for molecularly targeted drugs. Gene candidates were validated using western blot, qPCR, soft agar assay. Ewing’s Sarcoma A673 ABL2i knockdown and scramble cell lines were generated, implanted into nude mice, and treated with SP-2577. Lastly, combinational drug treatments using FDA-approved inhibitors with SP-2577 were tested in 2D and 3D cultures. Results: CRISPR druggable library screen identified that downregulation of several genes resulted in increased SP-2577 cytotoxicity in multiple ES cell lines. Importantly, ABL2 knockdown was found to have a significant synergy in combination with SP-2577 treatment, both in vitro and in vivo. siRNA knockdown of ABL2 in combination with SP-2577 treatment revealed an increase in JNK signaling and Cleaved Caspases-3 activation with decreased Src phosphorylation. This data suggests that downregulation of ABL2 in combination with SP-2577 treatment promotes activation of apoptosis pathways in ES. In addition, combination treatment of SP-2577 with drugs targeting ABL2 pathway showed high synergy in vitro. Conclusion: This study highlights the potential for a combination treatment of SP-2577 with ABL2 inhibitors in ES patients. Further, our observations support the capability of arrayed CRISPR screening to identify new therapeutic strategies in ES. Citation Format: Samuel Sampson, Trason Thode, Kevin Drenner, Ryan Rodriguez Del Villar, Annika Ozols, Mohan Kaadige, Alexis Weston, Serina Ng, Tithi Ghosh Halder, Shelby Rheinschmidt, David Anderson, Kevin Holden, Raffaella Soldi, Sunil Sharma. Synergistic drug combination of LSD1 and ABL2 inhibitors for Ewing's sarcoma identified in an arrayed CRISPR screen approach [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 1048.

Furfural Produces Dose-Dependent Attenuating Effects on Ethanol-Induced Toxicity in the Liver.

Background: Alcohol-associated liver disease (ALD) increases the health burden worldwide, but effective drugs to prevent ALD are lacking. Furfural is a small molecule that can limit alcohol production in microorganisms and may have the capacity to attenuate ethanol-induced toxicity. Methods: Human HepG2 cells were incubated with ethanol and furfural, and cell viability, NAD+/NADH ratio, and mitochondrial function assays were performed. RNA sequencing (RNA-seq) data were used to annotate enriched pathways, and these findings were confirmed by reverse transcription-quantitative PCR (RT–qPCR) and Western blotting. C57BL/6J mice were fed a Lieber-DeCarli liquid diet. After 4 weeks, biochemical analysis of mouse serum and histological analysis of mouse livers were performed. Results: Different concentrations of furfural exerted different effects on mitochondria: low-dose furfural reduced reactive oxygen species (ROS) production, maintained mitochondrial transmembrane potential, and inhibited apoptosis pathway activation, while high-dose furfural led to the opposite effects. In mice, furfural mitigated transaminase increases and attenuated the lipid metabolism disorder that had been induced by ethanol. Conclusion: Low-dose furfural reduced ethanol-induced toxicity in the liver. Consuming food or beverages containing the appropriate level of furfural when drinking alcohol may be a convenient and useful way to prevent ALD.

Cineole alleviates the BPA-inhibited NETs formation by regulating the p38 pathway-mediated programmed cell death

Bisphenol A (BPA) is an endocrine disruptor, that can cause immune dysfunction. Cineole (CIN) has that effect of regulating immune function and resist oxidation. Neutrophil extracellular traps (NETs) are one of the ways to resist pathogen invasion. In order to explore the effects of BPA and CIN on the release of chicken NETs and the antagonistic effect of CIN, take chicken peripheral blood neutrophils as object of study, grouping as NC, CIN, BPA + CIN and BPA. SEM, flow cytometry, RT-PCR, Western-blot and other methods were used to detect related indicators. The results showed that BPA inhibited the activities of GPX, SOD and CAT, increased the contents of MDA and NO, increased the activity of iNOS. BPA exposure inhibited the expression of myeloperoxidase (MPO), neutrophil elastase (NE) and histone, and inhibited the release of NETs. BPA activated downstream apoptosis and necroptosis through the p38 mitogen-activated protein kinase (p38-MAPK) pathway, which increased the expression of cytochrome C (CytC), bcl-2 associated K protein gene (bak), cysteinyl aspartate specific proteinase 3 (caspase-3), cysteinyl aspartate specific proteinase 9 (caspase-9), receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 1 (RIPK3) and mixed lineage kinase domain-like protein (MLKL), decreased the expression of B-cell lymphoma-2 (bcl-2). However, the co-exposure of CIN and BPA partially recovered the release of NETs, alleviated BPA-induced oxidative stress, and inhibited the activation of p38-MAPK pathway, necroptosis, and mitochondrial apoptosis pathway. These results indicated that CIN modulated p38 pathway alleviated BPA-induced neutrophil necroptosis and apoptosis, and increased NETs formation.

Donor-acceptor structured photothermal COFs for enhanced starvation therapy

Targeted delivery of adequate glucose oxidase (GOX) into tumor cells by covalent organic frameworks (COFs) remains a great challenge due to their insolubility and easy aggregation. Herein, an effective strategy to synthesize nanosized donor–acceptor (D-A) structured COFs with tunable sizes, long-time water-dispersibility and special selectivity was developed. Designed D-A structure provided pathways and channels for effective charge carriers transport, and this endowed COFs with excellent photothermal property to greatly enhance starvation therapy efficiency. Large surface area and porosity in the cross-linked crystal allowed high GOX loading capacity, and further surface modification with biomolecules made it possess colloid-like water stability and targeted selectivity. All these good properties guaranteed much better in vivo and in vitro tumor ablation than single therapy, and outstanding anti-migration results were also achieved. Detailed cell apoptosis study indicated the combined therapy effectively produced reactive oxygen species, and apoptosis was induced by endogenous mitochondrial apoptosis pathway and Caspase-3 activation process, finally making cancer cells more sensitive to inadequate energy supply and high temperature. This work developed a new strategy to synthesize D-A structured photothermal COFs with good dispersibility and greatly enhance starvation therapy without any additional use of chemical drugs.

Photothermolysis Mediated by Gold Nanorods Conjugated with Epidermal Growth Factor Receptor (EGFR) Monoclonal Antibody Induces Apoptosis via the Mitochondrial Apoptosis Pathway in Laryngeal Squamous Cell Cancer.

Gold nanorods (AuNRs) have unique optical properties and biological affinity and can be used to treat tumors when conjugated with other protein molecules. Our previous studies have shown that EGFR monoclonal antibody (EGFRmAb)-modified AuNRs exert strong antitumor activity in vitro by inducing apoptosis. In this study, we tested the effects of EGFRmAb-modified AuNRs on laryngeal squamous cell cancer (LSCC) in vitro and in vivo. The in vitro results showed that EGFRmAb-modified AuNRs inhibited NP-69, BEAS-2B and Hep-2 cell growth and induced mitochondria-dependent apoptosis. The mitochondrial membrane potential was reduced, leading to the release of cytochrome C (Cyt C) and consequent activation of the intrinsic mitochondrial apoptosis pathway. Moreover, we observed that the occurrence of mitochondrial apoptosis is related to the destruction of the lysosome-mitochondria axis. To verify the effects in vivo, we also established a laryngeal tumor model in nude mice by subcutaneous transplantation. In model mice treated with EGFRmAb-modified AuNRs and irradiated with an NIR laser, tumor cell apoptosis and tumor growth were inhibited. These results suggest that EGFRmAb-modified AuNRs induced apoptosis through the intrinsic mitochondrial apoptotic pathway and are a potential candidate for cancer therapy.

Krill Oil Attenuates Inflammation in Monosodium Iodoacetate-Induced Osteoarthritic Rats, SW982 Synovial Cell Line, and Primary Chondrocytes.

The aim of this study was to investigate the effects of krill oil (FJH-KO) in monoiodoacetate (MIA)-induced osteoarthritis in rat models, and H2O2- or lipopolysaccharide (LPS)-treated primary chondrocytes and the SW982 synovial cell line. We found that 150 mg/kg b.w. FJH-KO supplementation increased running speed, stride, and foot pressure in MIA-induced osteoarthritic rats. In the H2O2-treated SW982 synovial cell line and primary chondrocytes, FJH-KO treatment prevented cell death and suppressed matrix degradation by increasing the levels of anabolic factors of cartilage tissue, including aggrecan, collagen type Ⅰ, collagen type Ⅱ, tissue inhibitors of metalloproteinase (TIMP)-1, and TIMP-3, and decreasing those of catabolic factors of cartilage tissue, including phosphorylation of Smad, MMP-3, and MMP-13. In addition, FJH-KO treatment suppressed the activation of inflammation and apoptosis pathways in the LPS-treated SW982 synovial cell line and primary chondrocytes. We suggest that FJH-KO supplementation may help prevent osteoarthritis progression because of its direct effects on inflammation and apoptosis of chondrocytes.

Characterization of chikusetsusaponin IV and V induced apoptosis in HepG2 cancer cells

Chikusetsusaponin IV and V (CsIV and CsV), two typical oleanolic acid saponins, are mainly derived from the rhizome of Panax japonicus C.A. Mey. To reveal the anti-cancer effect of CsIV and CsV on liver cancer cells, human hepatic cancer cell lines (HepG2) were exposed to these saponins, and various physiological responses of HepG2 were investigated. HepG2 cells were treated with CsIV, CsV and 5-fluorouracil (5-FU). Cell proliferation was measured by CCK-8 assay. The cell cycle arrest, cell apoptosis and intracellular Ca2+ levels were respectively identified by flow cytometry. The mitochondrial membrane potential was detected by fluorescence microscopy. And, the levels of apoptosis-related proteins were analyzed by western blotting. Both CsIV and CsV were demonstrated to inhibit cell viability, and induce cell cycle arrest and apoptosis of HepG2 in a dose-dependent manner. They also enhanced the intracellular Ca2+ level and decreased the mitochondrial membrane potential in HepG2 cells. Furthermore, p53 and p21 were found up-regulated in HepG2 cells treated by CsIV and CsV. The apoptotic proteins, bax, cytochrome c, cleaved caspase-3/-9, were all found activated in HepG2 cells after CsIV and CsV treatment. The anti-apoptotic protein, bcl-2, was significantly down-regulated in all treated HepG2 cells. Our data demonstrated that CsIV and CsV exerted significant cytotoxic effects on HepG2 cells without affecting normal liver cells. And, these chikusetsusaponins, especially for CsIV, showed a potent effect on promoting cell apoptosis in HepG2 cells, which was associated with the activation of p53-mediated apoptosis pathway.

Combining selinexor with alisertib to target the p53 pathway in neuroblastoma.

Neuroblastoma accounts for 15% of cancer-related deaths in children, highlighting an unmet need for novel therapies. Selinexor is a small molecule inhibitor of XPO1. XPO1 shuffles cargo proteins with a nuclear export sequence from the nucleus to the cytosol, many of which are essential for cancer growth and cell maintenance. We systematically tested the effect of selinexor against neuroblastoma cells in vitro and in vivo and used an advanced proteomic and phosphoproteomic screening approach to interrogate unknown mechanisms of action. We found that selinexor induced its cytotoxic effects in neuroblastoma through the predominantly nuclear accumulation of p53 and global activation of apoptosis pathways. Selinexor also induced p53 phosphorylation at site S315, which is one initiating step for p53 degradation. Since this phosphorylation step is undertaken mostly by aurora kinase A (AURKA), we used the clinically available AURKA inhibitor, alisertib, and found p53-mediated lethality could be further augmented in three orthotopic xenograft mouse models. These findings suggest a potential therapeutic benefit using selinexor and alisertib to synergistically increase p53-mediated cytotoxicity of high-risk neuroblastoma.