Extrinsic Pathway Research Articles

The Association of Death Receptors and TGF-β1 Expression in Urothelial Bladder Cancer and Their Prognostic Significance.

Death receptor signalization that triggers the extrinsic apoptotic pathway and TGF-β1 have important roles in urothelial carcinogenesis, with a complex interplay between them. The aim of this research was to assess the association of death receptors DR4, DR5, and FAS as well as TGF-β1 immunohistochemical expression with the clinicopathological characteristics of urothelial bladder cancer (UBC) and to evaluate their prognostic significance. The decrease or loss of death receptors' expression was significantly associated with muscle-invasive tumors, while non-invasive UBC often retains the expression of death receptors, which are mutually strongly linked. High DR4 expression is a marker of low-grade tumors and UBC associated with exposition to known carcinogens. Conversely, TGF-β1 was significantly associated with high tumor grade and advanced stage. High expression of DR4 and FAS indicates longer overall survival. High TGF-β1 signifies an inferior outcome and is an independent predictor of adverse prognosis in UBC patients. This study reveals the expression profile of death receptors in UBC and their possible interconnection with TGF-β1 and indicates independent prognostic significance of high FAS and TGF-β1 expression in UBC, which may contribute to deciphering the enigma of UBC heterogeneity in light of the rapid development of novel and effective therapeutic approaches, including targeting of the TRAIL-induced apoptotic pathway.

Point-of-care viscoelastic coagulation monitor parameters in Amazon parrots (Amazona spp.).

To evaluate the feasibility of the point-of-care viscoelastic coagulation monitor (VCM) in Amazon parrots (Amazona spp.) and describe the parameters with fresh whole-blood samples in healthy Amazon parrots. A total of 18 Amazon parrots were enrolled. Physical examinations, a CBC, and a biochemistry profile, including bile acids, were performed on all parrots. VCM tracings were obtained at the time of venipuncture for baseline laboratory work. The median clot time was 2102seconds (range: 38.6-3599s), median clot formation time was 929seconds (range: 21.4-1711s), median alpha angle was 20 (range: 6-67), and the median maximum clot formation was 8.5 (range: 0-36). The median lysis index at 30minutes (LI30) was 100 (range: 98-100), and the median lysis index at 45minutes (LI45) was 100 (range: 90-100). Of 18 samples, alpha angles were not reported in 7 samples, LI30 was not reported in 10 samples, and LI45 was not reported in 12 samples. Of the qualitative curves, 6 reflected normal mammalian curves, and the remainder were consistent with a hypocoaguable state. The results were markedly variable, with the majority of VCM tracings being hypocoagulable in comparison with reference intervals established for dogs and cats. Using these protocols, the VCM is not reliable in Amazon parrots. Future areas of investigation include altering the temperature during sample analysis, the use of activators, or an exchange of clotting reagents for an extrinsic pathway activator, which may contribute to the success of this device in avian species.

Network pharmacology and molecular docking technology for exploring the effect and mechanism of high-dose vitamin c on ferroptosis of tumor cells: A review.

To investigate the mechanism by which high-dose vitamin C (HVC) promotes ferroptosis in tumor cells via network pharmacology, vitamin C-related and ferroptosis-related targets were obtained from the PharmMapper and GeneCards databases, respectively, and their common targets were compared using the Venn diagram. Common targets were imported into the STRING database for protein-protein interaction analysis, and core targets were defined. Core targets were enriched for Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways using the R language packages. A map of the core target-based interaction network and a map of the mechanism by which HVC regulates ferroptosis were constructed. A total of 238 vitamin C-related and 721 ferroptosis-related targets were identified, of which 21 targets were common to both. Furthermore, ALDOA, AHCY, LDHB, HSPA8, LGALS3, and GSTP1 were identified as core targets. GO enrichment analysis suggested that the main biological processes included the extrinsic apoptotic signaling pathway and pyruvate metabolic process. KEGG enrichment analysis suggested that HVC regulates ferroptosis mainly through the amino acid and carbohydrate metabolic pathways. The targets were validated by molecular docking. In conclusion, HVC may promote ferroptosis in tumor cells by regulating metabolic pathways, and there is a synergistic effect between HVC and type I ferroptosis inducers. Glycolysis-dependent tumors may be beneficial for HVC therapy. Our study provides a reference for further clinical studies on HVC antitumor therapy.

Combination therapy of cisplatin and green silver nanoparticles enhances cytotoxicity and apoptosis in breast cancer cells

Cisplatin is one of the first-line drugs for the treatment of breast cancer and is known for its ability to disrupt cancer cell DNA. However, cisplatin chemotherapy carries side effects and the risk of drug resistance. A strategy to improve its anti-cancer efficacy while reducing its negative effects on health is to leverage the synergistic potential of natural molecules with cisplatin. In this study, we explored combination therapy using cisplatin along with biosynthesized silver nanoparticles (Ag NPs) to enhance apoptosis induction in MCF-7 breast cancer cells while reducing cisplatin resistance. The biosynthesized Ag NPs, derived from Acacia Luciana flower extracts, possess active molecules that effectively inhibit MCF-7 cells. Concurrent administration of cisplatin and Ag NPs resulted in a notable decrease in the IC50 value – approximately 22 – 26 times lower compared to individual treatments of free Ag NPs and cisplatin, respectively. Furthermore, the combination therapy significantly increased the BAK1/BCLX ratio by 162-fold compared to the control, while the cisplatin alone failed to activate intrinsic apoptosis pathway. In addition, the expression level of the CASP3 gene, indicative of the extrinsic pathway of apoptosis, increased approximately 273 times compared to free cisplatin (CASP3 expression = 3.5). Notably, the combination therapy also reduced the expression of the AKT1 gene, associated with cell survival and treatment resistance, when compared to free cisplatin (1.87 vs. 4.488). In conclusion, our findings proved that combination therapy effectively enhances apoptosis induction by cisplatin while reducing drug resistance.

Carbon nanotubes conjugated with cisplatin activate different apoptosis signaling pathways in 2D and 3D-spheroid triple-negative breast cancer cell cultures: a comparative study

The type of experimental model for the in vitro testing of drug formulations efficiency represents an important tool in cancer biology, with great attention being granted to three-dimensional (3D) cultures as these offer a closer approximation of the clinical sensitivity of drugs. In this study, the effects induced by carboxyl-functionalized single-walled carbon nanotubes complexed with cisplatin (SWCNT–COOH–CDDP) and free components (SWCNT–COOH and CDDP) were compared between conventional 2D- and 3D-spheroid cultures of human breast cancer cells. The 2D and 3D breast cancer cultures were exposed to various doses of SWCNT–COOH (0.25–2 μg/mL), CDDP (0.158–1.26 μg/mL) and the same doses of SWNCT–COOH–CDDP complex for 24 and 48 h. The anti-tumor activity, including modulation of cell viability, oxidative stress, proliferation, apoptosis, and invasion potential, was explored by spectrophotometric and fluorometric methods, immunoblotting, optical and fluorescence microscopy. The SWCNT–COOH–CDDP complex proved to have high anti-cancer efficiency on 2D and 3D cultures by inhibiting cell proliferation and activating cell death. A dose of 0.632 μg/mL complex triggered different pathways of apoptosis in 2D and 3D cultures, by intrinsic, extrinsic, and reticulum endoplasmic pathways. Overall, the 2D cultures showed higher susceptibility to the action of complex compared to 3D cultures and SWCNT–COOH–CDDP proved enhanced anti-tumoral activity compared to free CDDP.

Platelets Induce Cell Apoptosis of Cardiac Cells via FasL after Acute Myocardial Infarction.

Acute myocardial infarction (AMI) is one of the leading causes of death worldwide. Cell apoptosis in the myocardium plays an important role in ischemia and reperfusion (I/R) injury, leading to cardiac damage and dysfunction. Platelets are major players in hemostasis and play a crucial role in vessel occlusion, inflammation, and cardiac remodeling after I/R. Here, we studied the impact of platelets on cell apoptosis in the myocardium using a close-chest mouse model of AMI. We found caspase-3-positive resident cardiac cells, while leukocytes were negative for caspase-3. Using two different mouse models of thrombocytopenia, we detected a significant reduction in caspase-3 positive cells in the infarct border zone after I/R injury. Further, we identified platelet FasL to induce cell apoptosis via the extrinsic pathway of Fas receptor activation of target cells. Mechanistically, hypoxia triggers platelet adhesion to FasR, suggesting that platelet-induced apoptosis is elevated after I/R. Platelet-specific FasL knock-out mice showed reduced Bax and Bcl2 expression, suggesting that platelets modulate the intrinsic and extrinsic pathways of apoptosis, leading to reduced infarct size after myocardial I/R injury. Thus, a new mechanism for how platelets contribute to tissue homeostasis after AMI was identified that should be validated in patients soon.

Arginine methylation of caspase-8 controls life/death decisions in extrinsic apoptotic networks

Procaspase-8 is a key mediator of death receptor (DR)-mediated pathways. Recently, the role of post-translational modifications (PTMs) of procaspase-8 in controlling cell death has received increasing attention. Here, using mass spectrometry screening, pharmacological inhibition and biochemical assays, we show that procaspase-8 can be targeted by the PRMT5/RIOK1/WD45 methylosome complex. Furthermore, two potential methylation sites of PRMT5 on procaspase-8, R233 and R435, were identified in silico. R233 and R435 are highly conserved in mammals and their point mutations are among the most common mutations of caspase-8 in cancer. The introduction of mutations at these positions resulted in inhibitory effects on CD95L-induced caspase-8 activity, effector caspase activation and apoptosis. In addition, we show that procaspase-8 can undergo symmetric di-methylation. Finally, the pharmacological inhibition of PRMT5 resulted in the inhibitory effects on caspase activity and apoptotic cell death. Taken together, we have unraveled the additional control checkpoint in procaspase-8 activation and the arginine methylation network in the extrinsic apoptosis pathway.

Anti-proliferation evaluation of new derivatives of indole-6-carboxylate ester as receptor tyrosine kinase inhibitors.

Aim: The main goal was to create two new groups of indole derivatives, hydrazine-1-carbothioamide (4a and 4b) and oxadiazole (5, and 6a-e) that target EGFR (4a, 4b, 5) or VEGFR-2 (6a-e). Materials & methods: The new derivatives were characterized using various spectroscopic techniques. Docking studies were used to investigate the binding patterns to EGFR/VEGFR-2, and the anti-proliferative properties were tested in vitro. Results: Compounds 4a (targeting EGFR) and 6c (targeting VEGFR-2) were the most effective cytotoxic agents, arresting cancer cells in the G2/M phase and inducing the extrinsic apoptosis pathway. Conclusion: The results of this study show that compounds 4a and 6c are promising cytotoxic compounds that inhibit the tyrosine kinase activity of EGFR and VEGFR-2, respectively.

P,p'-DDT induces apoptosis in human endometrial stromal cells via the PI3K/AKT pathway and oxidative stress.

Bis-[4-chlorophenyl]-1,1,1-trichloroethane (DDT), one of the most widely used synthetic pesticides, is an endocrine-disrupting chemical with the potential to interfere with the human reproductive system. The effects of DDT and one of its metabolites, p,p'-DDT, on human endometrial stromal cells (ESCs) and health outcomes remain unknown. In this study, we investigated whether p,p'-DDT induces an imbalance in cell proliferation and apoptosis in human ESCs via oxidative stress. We assessed apoptosis in ESCs by quantifying the expression of markers associated with both intrinsic and extrinsic pathways. Additionally, we measured levels of reactive oxygen species (ROS), antioxidant enzyme activity, and estrogen receptors (ERs). We also examined changes in signaling involving nuclear factor kappa-light-chain-enhancer of activated B cells. Following treatment with 1,000 pg/mL of p,p'-DDT, we observed an increase in Bax expression, a decrease in Bcl-2 expression, and increases in the expression of caspases 3, 6, and 8. We also noted a rise in the generation of ROS and a reduction in glutathione peroxidase expression after treatment with p,p'-DDT. Additionally, p,p'-DDT treatment led to changes in ER expression and increases in the protein levels of phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (phospho-AKT), and phospho-extracellular signal-regulated kinase (phospho-ERK). p,p'-DDT was found to induce apoptosis in human ESCs through oxidative stress and an ER-mediated pathway. The activation of the PI3K/AKT and ERK pathways could represent potential mechanisms by which p,p'-DDT prompts apoptosis in human ESCs and may be linked to endometrial pathologies.

Evaluating anthelmintic, anti-platelet, and anti-coagulant activities, and identifying the bioactive phytochemicals of Amaranthus blitum L.

BackgroundHighlighting affordable alternative crops that are rich in bioactive phytoconstituents is essential for advancing nutrition and ensuring food security. Amaranthus blitum L. (AB) stands out as one such crop with a traditional history of being used to treat intestinal disorders, roundworm infections, and hemorrhage. This study aimed to evaluate the anthelmintic and hematologic activities across various extracts of AB and investigate the phytoconstituents responsible for these activities.MethodsIn vitro anthelmintic activity against Trichinella spiralis was evaluated in terms of larval viability reduction. The anti-platelet activities were assessed based on the inhibitory effect against induced platelet aggregation. Further, effects on the extrinsic pathway, the intrinsic pathway, and the ultimate common stage of blood coagulation, were monitored through measuring blood coagulation parameters: prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT), respectively. The structures of isolated compounds were elucidated by spectroscopic analysis.ResultsInterestingly, a previously undescribed compound (19), N-(cis-p-coumaroyl)-ʟ-tryptophan, was isolated and identified along with 21 known compounds. Significant in vitro larvicidal activities were demonstrated by the investigated AB extracts at 1 mg/mL. Among tested compounds, compound 18 (rutin) displayed the highest larvicidal activity. Moreover, compounds 19 and 20 (N-(trans-p-coumaroyl)-ʟ-tryptophan) induced complete larval death within 48 h. The crude extract exhibited the minimal platelet aggregation of 43.42 ± 11.69%, compared with 76.22 ± 14.34% in the control plasma. Additionally, the crude extract and two compounds 19 and 20 significantly inhibited the extrinsic coagulation pathway.ConclusionsThese findings extend awareness about the nutritional value of AB as a food, with thrombosis-preventing capabilities and introducing a promising source for new anthelmintic and anticoagulant agents.

Discovery of (−)-epigallocatechin gallate, a novel olfactory receptor 2AT4 agonist that regulates proliferation and apoptosis in leukemia cells

Olfactory receptors (ORs), the largest family of G protein-coupled receptors (GPCRs), are ectopically expressed in cancer cells and are involved in cellular physiological processes, but their function as anticancer targets is still potential. OR2AT4 is expressed in leukemia cells, influencing the proliferation and apoptosis, yet the limited number of known OR2AT4 agonists makes it challenging to fully generalize the receptor's function. In this study, we aimed to identify new ligands for OR2AT4 and to investigate their functions and mechanisms in K562 leukemia cells. After producing the recombinant OR2AT4 protein, immobilizing it on a surface plasmon resonance chip, and conducting screening to confirm binding activity using 258 chemicals, five novel OR2AT4 ligands were discovered. As a result of examining changes in intracellular calcium by five ligands in OR2AT4-expressing cells and K562 cells, (−)-epigallocatechin gallate (EGCG) was identified as an OR2AT4 agonist in both cells. EGCG reduced the viability of K562 cells and induced apoptosis in K562 cells. EGCG increased the expression of cleaved caspase 3/8 and had no effect on the expression of Bax and Bcl-2, indicating that it induced apoptosis through the extrinsic pathway. Additionally, the initiation of the extrinsic apoptosis pathway in EGCG-induced K562 cells was due to the activation of OR2AT4, using an OR2AT4 antagonist. This study highlights the potential of EGCG as an anti-cancer agent against leukemia and OR2AT4 as a target, making it a new anti-cancer drug.

Looking for ALPS: The value of a combined assessment of biochemical markers.

Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune disorder caused by defect of the extrinsic apoptotic pathway. The current diagnostic criteria combine clinical features and typical biomarkers but have not been the object of clear international consensus. We conducted a retrospective study on pediatric patients who were investigated for autoimmune cytopenia and/or lymphoproliferation at the CHU Sainte-Justine Hospital over 10 years. Patients were screened using the combination of TCRαβ+ CD4- CD8- "double negative" (DN) T cells and soluble plasmatic FAS ligand (sFASL). Among the 398 tested patients, the median sFASL and DN T cells were 200 ng/mL and 1.8% of TCRαβ+ T cells, respectively. sFASL was highly correlated with vitamin B12 levels. We identified five patients diagnosed with ALPS for whose sFASL and vitamin B12 levels were the more discriminating biomarkers. While ALPS diagnostic criteria had high sensibility, their predictive value remained low. sFASL level can efficiently discriminate patients with ALPS when using the appropriate thresholds. Our study highlights the need for an international consensus to redefine the place and threshold of biological biomarkers for ALPS diagnosis.

Macromolecules with predominant β-pleated sheet proteins in extracellular vesicles released from Raphanus sativus L. var. caudatus Alef microgreens induce DNA damage-mediated apoptosis in HCT116 colon cancer cells

Plant-derived bioactive macromolecules (i.e., proteins, lipids, and nucleic acids) were prepared as extracellular vesicles (EVs). Plant-derived EVs are gaining pharmaceutical research interest because of their bioactive components and delivery properties. The spherical nanosized EVs derived from Raphanus sativus L. var. caudatus Alef microgreens previously showed antiproliferative activity in HCT116 colon cancer cells from macromolecular compositions (predominantly proteins). To understand the mechanism of action, the biological activity studies, i.e., antiproliferation, cellular biochemical changes, DNA conformational changes, DNA damage, apoptotic nuclear morphological changes, apoptosis induction, and apoptotic pathways, were determined by neutral red uptake assay, synchrotron radiation-based Fourier transform infrared microspectroscopy, circular dichroism spectroscopy, comet assay, 4′,6-diamidino-2-phenylindole (DAPI) staining, flow cytometry, and caspase activity assay, respectively. EVs inhibited HCT116 cell growth in concentration- and time-dependent manners, with a half-maximal inhibitory concentration of 675.4 ± 33.8 μg/ml at 48 h and a selectivity index of 1.5 ± 0.076. HCT116 treated with EVs mainly changed the cellular biochemical compositions in the nucleic acids and carbohydrates region. The DNA damage caused no changes in DNA conformation. The apoptotic nuclear morphological changes were associated with the increased apoptotic cell population. The apoptotic cell death was induced by both extrinsic and intrinsic pathways. EVs have potential as antiproliferative bioparticles.

Intrinsic and Extrinsic Apoptotic Pathways May Dictate In Vitro Fertilization Outcomes in Women of Advanced Maternal Age [ID 2683425

INTRODUCTION: Women of advanced maternal age (AMA) (≥35 years) are at an increased risk for a decline in ovarian reserve and have a decreased rate of live birth after one cycle of in vitro fertilization (IVF). Current practice recommends preimplantation genetic testing for aneuploidy (PGT-A) to women of AMA to increase live birth outcomes. However, recent studies have found that conventional IVF was non-inferior to PGT-A. Identifying additional biomarkers may provide another metric for selecting the most viable embryo. METHODS: Blastocoel fluid-conditioned media was collected from day 5 IVF embryos that underwent PGT-A. RNA was purified from pooled blastocoel fluid samples from four groups based on implantation status and maternal age, and then cDNA was synthesized. RT-qPCR was used to assess expression of CASP3, CASP7, CASP8, and BCL2L12 levels in a total of 64 embryos. RESULTS: CASP3 and CASP7 expression were higher in the media from IVF embryos with positive implantation outcomes from patients aged less than 35 years. There was increased CASP8 expression in media from IVF embryos from AMA patients regardless of implantation status. CONCLUSION: Preliminary analysis suggests increased CASP8 expression is associated with successful implantation outcomes in AMA patients. This suggests that CASP8 could be activated in preimplantation embryos as an aging response as found in AMA media samples. One explanation is that CASP8 is associated with aneuploidy in the AMA samples. Further research will determine whether expression and activation of CASP8 are required for positive implantation outcomes and be used as an additional embryo selection tool for other AMA patients utilizing IVF.

MORIN ATTENUATES INFLAMMASOME-DEPENDENT MYOCARDIAL INJURY IN DIABETIC RATS: MODULATION OF THE SIGNALING PATHWAY

Objective: Diabetes is a risk factor that predisposes to atherosclerotic cardiovascular diseases. The risk of myocardial infarction in diabetes is higher and this warrants investigation into exploring new drugs that can attenuate myocardial injury. In diabetes, high blood glucose levels lead to vascular inflammation which accelerates atherosclerosis. Morin, a plant flavonoid, was found effective for septic shock, cancer, cardiovascular, and renal complications and acts as an agonist for insulin secretion. This study aims to evaluate the mechanism involved in the cardioprotective action of Morin in diabetic rats. Design and method: In male Wistar rats, streptozotocin (70 mg/kg; i.p.) was administered to induce diabetes. Diabetes-confirmed rats (blood glucose >400mg/dl) were divided into 4 groups (n=8), i.e., Diabetic-control; Diabetes+Isoproterenol (ISO); Diabetes+ISO+Morin and Diabetes+Morin, in addition to this, one control group was also included in the study. Morin was orally administered at the dose of 40 mg/kg for 28 days and on the 27th and 28th day of morin administration, ISO was administered to designate groups at the dose of 85mg/kg s.c., to induce myocardial injury. After 24 hours of the second dose of ISO administration, the rat was sacrificed after collection of blood, and heart was excised to do the biochemical, histopathological and molecular investigations. Results: ROS rush following ISO administration leads to the activation of the intrinsic and extrinsic pathways of apoptosis. Morin significantly reduced oxidative stress, cardiac injury markers levels, inflammation, and apoptosis. In addition, it also reduced serum insulin and blood glucose levels. Histopathology showed cardio-protection with morin. Akt/eNOS, Nrf2/HO-1, MAPK signaling pathways, and Insulin signal transduction pathways were positively modulated by Morin pre-treatment. It also significantly modulated NLRP3 inflammasome formation. Conclusions: Morin attenuated oxidative stress and inflammation and also modified the activity of various molecular pathways to mitigate cardiomyocyte damage during ISO-induced MI in diabetic rats.

Comparison of 177Lu-octreotate and 177Lu-octreotide for treatment in human neuroblastoma-bearing mice

BackgroundPatients with high-risk neuroblastoma (NB) have a 5-year event-free survival of less than 50 %, and novel and improved treatment options are needed. Radiolabeled somatostatin analogs (SSTAs) could be a treatment option. The aims of this work were to compare the biodistribution and the therapeutic effects of 177Lu-octreotate and 177Lu-octreotide in mice bearing the human CLB-BAR NB cell line, and to evaluate their regulatory effects on apoptosis-related genes. MethodsThe biodistribution of 177Lu-octreotide in mice bearing CLB-BAR tumors was studied at 1, 24, and 168 h after administration, and the absorbed dose was estimated to tumor and normal tissues. Further, animals were administered different amounts of 177Lu-octreotate or 177Lu-octreotide. Tumor volume was measured over time and compared to a control group given saline. RNA was extracted from tumors, and the expression of 84 selected genes involved in apoptosis was quantified with qPCR. ResultsThe activity concentration was generally lower in most tissues for 177Lu-octreotide compared to 177Lu-octreotate. Mean absorbed dose per administered activity to tumor after injection of 1.5 MBq and 15 MBq was 0.74 and 0.03 Gy/MBq for 177Lu-octreotide and 2.9 and 0.45 Gy/MBq for 177Lu-octreotate, respectively. 177Lu-octreotide treatment resulted in statistically significant differences compared to controls. Fractionated administration led to a higher survival fraction than after a single administration. The pro-apoptotic genes TNSFS8, TNSFS10, and TRADD were regulated after administration with 177Lu-octreotate. Treatment with 177Lu-octreotide yielded regulation of the pro-apoptotic genes CASP5 and TRADD, and of the anti-apoptotic gene IL10 as well as the apoptosis-related gene TNF. Conclusion177Lu-octreotide gave somewhat better anti-tumor effects than 177Lu-octreotate. The similar effect observed in the treated groups with 177Lu-octreotate suggests saturation of the somatostatin receptors. Pronounced anti-tumor effects following fractionated administration merited receptor saturation as an explanation. The gene expression analyses suggest apoptosis activation through the extrinsic pathway for both radiopharmaceuticals.

Marine-derived antimicrobial peptide piscidin-1 triggers extrinsic and intrinsic apoptosis in oral squamous cell carcinoma through reactive oxygen species production and inhibits angiogenesis

Cancer of the head and neck encompasses a wide range of cancers, including oral and oropharyngeal cancers. Oral cancer is often diagnosed at advanced stages and has a dismal prognosis. Piscidin-1, a marine antimicrobial peptide (AMP) containing approximately 22 amino acids, also exhibits significant anticancer properties. We investigated the possible anti-oral cancer effects of piscidin-1 and clarified the mechanisms underlying these effects. We treated the oral squamous cell carcinoma cell lines OC2 and SCC4 with piscidin-1. Cell viability and the expression of different hallmark apoptotic molecules, including reactive oxygen species (ROS), were tested using the appropriate MTT assay, flow cytometry and western blotting assays, and human umbilical vein endothelial cell (HUVEC) wound healing, migration, and tube formation (angiogenesis) assays. Piscidin-1 increases cleaved caspase 3 levels to induce apoptosis. Piscidin-1 also increases ROS levels and intensifies oxidative stress in the endoplasmic reticulum and mitochondria, causing mitochondrial dysfunction. Additionally, it decreases the oxygen consumption rates and activity of mitochondrial complexes I–V. As expected, the antioxidants MitoTEMPOL and N-acetylcysteine reduce piscidin-1–induced ROS generation and intracellular calcium accumulation. Piscidin-1 also inhibits matrix metalloproteinase (MMP)-2/-9 expression in HUVECs, affecting migration and tube formation angiogenesis. We demonstrated that piscidin-1 can promote apoptosis via both intrinsic and extrinsic apoptotic pathways and findings indicate that piscidin-1 has anti-proliferative and anti-angiogenic properties in oral cancer treatment. Our study on piscidin-1 thus provides a basis for future translational anti-oral cancer drug research and a new theoretical approach for anti-oral cancer clinical research.

Enrichment and Evaluation of Antitumor Properties of Total Flavonoids from Juglans mandshurica Maxim.

Flavonoids are important secondary metabolites found in Juglans mandshurica Maxim., which is a precious reservoir of bioactive substances in China. To explore the antitumor actions of flavonoids (JMFs) from the waste branches of J. mandshurica, the following optimized purification parameters of JMFs by macroporous resins were first obtained. The loading concentration, flow rate, and loading volume of raw flavonoid extracts were 1.4 mg/mL, 2.4 BV/h, and 5 BV, respectively, and for desorption, 60% ethanol (4 BV) was selected to elute JMFs-loaded AB-8 resin at a flow rate of 2.4 BV/h. This adsorption behavior can be explained by the pseudo-second-order kinetic model and Langmuir isotherm model. Subsequently, JMFs were identified using Fourier transform infrared combined with high-performance liquid chromatography and tandem mass spectrometry, and a total of 156 flavonoids were identified. Furthermore, the inhibitory potential of JMFs on the proliferation, migration, and invasion of HepG2 cells was demonstrated. The results also show that exposure to JMFs induced apoptotic cell death, which might be associated with extrinsic and intrinsic pathways. Additionally, flow cytometry detection found that JMFs exposure triggered S phase arrest and the generation of reactive oxygen species in HepG2 cells. These findings suggest that the JMFs purified in this study represent great potential for the treatment of liver cancer.

SARS-CoV-2-ORF3a variant Q57H reduces its pro-apoptotic activity in host cells

Background Mutations in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can enhance its pathogenicity by affecting its transmissibility, disease severity, and overall mortality in human populations. In addition to mutations within the coding region of SARS-CoV-2 structural proteins, there have been reports of mutations in other SARS-CoV-2 proteins that affect virulence, such as open reading frame 3a (ORF3a), which is involved in viral replication. The expression of ORF3a in host cells activates cell death signaling, leading to tissue damage, which affects the severity of COVID-19. The ORF3a-Q57H variant is the most frequent and recurrent variant of ORF3a and is likely associated with increased transmissibility but lower mortality in the 4th epidemic wave of COVID-19 in Hong Kong. Computational structural modeling predicted that the Q57H variant destabilizes the protein structure of ORF3a, which may result in reduced protein expression in human cells. However, it is still unknown how this mutation affects ORF3a protein function and, if so, whether it can change the severity of host cell damage. Methods Plasmids carrying SARS-CoV-2-ORF3a from Wuhan-Hu-1 strain (i.e., wild-type; WT) and its variant Q57H were transiently transfected into HEK293T cells and used for biochemical and cell biological assays. Results SARS-CoV-2-ORF3a-Q57H variant exhibits higher protein expression than WT, but ORF3a-Q57H expression results in less apoptosis in host cells compared to WT via lower activation of the extrinsic apoptotic pathway. Conclusion The relatively mild phenotype of the SARS-CoV-2-ORF3a-Q57H variant may result from alterations to ORF3a function by this mutation, rather than its protein expression levels in host cells.

Synthesis, docking study, and antitumor evaluation of benzamides and oxadiazole derivatives of 3-phenoxybenzoic acid as VEGFR-2 inhibitors.

Current chemotherapeutic agents have several limitations, including lack of selectivity, the development of undesirable side effects, and chemoresistance. As a result, there is an unmet need for the development of novel small molecules with minimal side effects and the ability to specifically target tumor cells. A new series of 3-phenoxybenzoic acid derivatives, including 1,3,4-oxadiazole derivatives (4a-d) and benzamides derivatives (5a-e) were synthesized; their chemical structures were confirmed by Fourier-transform infrared spectroscopy, 1H nuclear magnetic resonance (NMR), 13C NMR, and mass spectra; and various physicochemical properties were determined. The antiproliferative activities of the new derivatives were evaluated by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Three compounds (4b, 4c, and 4d) exhibited cytotoxicity against two of the three cell lines tested, five compounds (3, 4a, 5a, 5b, and 5e) were toxic to one cell line, while two compounds (5c and 5d) were not cytotoxic to any of the three cell lines tested in the current study. Based on docking scores, MTT assay findings, and vascular endothelial growth factor receptor 2 (VEGFR-2) kinase activity data, Compound 4d was selected for further biological investigation. Flow cytometry was used to determine the mode of cell death (apoptosis vs. necrosis) and the effect on cell cycle progression. Compound 4d arrested HepG2 hepatocellular carcinoma cells in the G2/M phase and activated both the intrinsic and extrinsic apoptosis pathways. In conclusion, Compound 4d has shown promising results for future research as a potent VEGFR-2 tyrosine kinase inhibitor.