Synthesis, cytotoxicity, anti-inflammatory, anti-metastatic and anti-oxidant activities of novel chalcones incorporating 2-phenoxy-N-arylacetamide and thiophene moieties: induction of apoptosis in MCF7 and HEP2 cells.

Eight Novel chalcones were synthesized and their structures were confirmed by different spectral tools. All the prepared compounds were subjected to SRB cytotoxic screening against several cancer cell lines. Compound 5c exerted the most promising effect against MCF7 and HEP2 cells with IC50 values of 9.5 and 12 µg/mL, respectively. Real-time PCR demonstrated the inhibitory effect of compound 5c on the expression level of Antigen kiel 67 (KI-67), Survivin, Interleukin-1beta (IL-1B), Interleukin-6 (IL-6), Cyclooxygenase-2 (COX-2) and Protein kinase B (AKT1) genes. Flow-cytometric analysis of the cell cycle indicated that compound 5c stopped the cell cycle at the G0/G1 and G2/M phases in MCF7 and HEP2 treated cells, respectively. ELISA assay showed that Caspase 8, Caspase 9, P53, BAX, and Glutathione (GSH) were extremely activated and Matrix metalloproteinase 2 (MMP2), Matrix metalloproteinase 9 (MMP9), BCL2, Malondialdehyde (MDA), and IL-6 were deactivated in 5c treated MCF7 and HEP2 cells. Wound healing revealed that chalcone 5c reduced the ability to close the scrape wound and decreased the number of migrating MCF7 and HEP2 cells compared to the untreated cells after 48 h. Theoretical molecular modeling against P53 cancer mutant Y220C and Bcl2 showed binding energies of -22.8 and -24.2 Kcal/mole, respectively, which confirmed our ELISA results.

Panoramic analysis of cell death patterns reveals prognostic and immune profiles of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) has been characterized by a low therapeutic response and poor prognosis. Currently, there are no reliable predictive models for HNSCC progression and therapeutic efficacy. This study explores the role of diverse patterns of cell death in tumor development, positing them as predictive factors of HNSCC prognosis. We utilized bulk transcriptome and single-cell transcriptome, align with clinical information from TCGA and GEO database, to analyze genes associated with 15 types of cell death and construct a cell death index (CDI) signature. The associations of CDI with tumor-infiltrating immune cells and immunotherapy-related biomarkers were also evaluated using various algorithms. The CDI signature emerged as a robust prognosis biomarker that could identify patients who can benefit potentially from immunotherapy, thus improving diagnostic accuracy and optimizing clinical decisions in HNSCC management. Notably, we discovered that CAAP1 deficiency not only induced apoptosis but also enhanced anti-tumor immunity, suggesting its potential as a target for clinical drug development.

Retinal response to systemic inflammation differs between sexes and neurons.

Background: Neurological dysfunction and glial activation are common in severe infections such as sepsis. There is a sexual dimorphism in the response to systemic inflammation in both patients and animal models, but there are few comparative studies. Here, we investigate the effect of systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) on the retina of male and female mice and determine whether antagonism of the NLRP3 inflammasome and the extrinsic pathway of apoptosis have protective effects on the retina. Methods: A single intraperitoneal injection of LPS (5 mg/kg) was administered to two months old C57BL/6J male and female mice. Retinas were examined longitudinally in vivo using electroretinography and spectral domain optical coherence tomography. Retinal ganglion cell (RGC) survival and microglial activation were analysed in flat-mounts. Retinal extracts were used for flow cytometric analysis of CD45 and CD11b positive cells. Matched plasma and retinal levels of proinflammatory cytokines were measured by ELISA. Retinal function and RGC survival were assessed in animals treated with P2X7R and TNFR1 antagonists alone or in combination. Results: In LPS-treated animals of both sexes, there was transient retinal dysfunction, loss of vision-forming but not non-vision forming RGCs, retinal swelling, microglial activation, cell infiltration, and increases in TNF and IL-1ß. Compared to females, males showed higher vision-forming RGC death, slower functional recovery, and overexpression of lymphotoxin alpha in their retinas. P2X7R and TNFR1 antagonism, alone or in combination, rescued vision-forming RGCs. P2X7R antagonism also rescued retinal function. Response to treatment was better in females than in males. Conclusions: Systemic LPS has neuronal and sex-specific adverse effects in the mouse retina, which are counteracted by targeting the NLRP3 inflammasome and the extrinsic pathway of apoptosis. Our results highlight the need to analyse males and females in preclinical studies of inflammatory diseases affecting the central nervous system.

TRAIL and its receptors in cardiac diseases.

Cardiovascular disease is a leading cause of death worldwide. Loss of cardiomyocytes that occurs during many types of damage to the heart such as ischemic injury and stress caused by pressure overload, diminishes cardiac function due to their limited regenerative capacity and promotes remodeling, which further damages the heart. Cardiomyocyte death occurs through two primary mechanisms, necrosis and apoptosis. Apoptosis is a highly regulated form of cell death that can occur through intrinsic (mitochondrial) or extrinsic (receptor mediated) pathways. Extrinsic apoptosis occurs through a subset of Tumor Necrosis Receptor (TNF) family receptors termed "Death Receptors." While some ligands for death receptors have been extensively studied in the heart, such as TNF-α, others have been virtually unstudied. One poorly characterized cardiac TNF related ligand is TNF-Related Apoptosis Inducing Ligand (TRAIL). TRAIL binds to two apoptosis-inducing receptors, Death Receptor (DR) 4 and DR5. There are also three decoy TRAIL receptors, Decoy Receptor (DcR) 1, DcR2 and osteoprotegerin (OPG). While TRAIL has been extensively studied in the cancer field due to its ability to selectively induce apoptosis in transformed cell types, emerging clinical evidence points towards a role for TRAIL and its receptors in cardiac pathology. This article will highlight our current understanding of TRAIL and its receptors in normal and pathological conditions in the heart.

A Dual Concentration-Tailored Cytokine-Chemo Nanosystem to Alleviate Multidrug Resistance and Redirect Balance of Cancer Proliferation and Apoptosis.

Background: Cancer multidrug resistance (MDR) is an important factor that severely affects the chemotherapeutic efficacy. Among various methods to bypass MDR, usage of cytokines, such as tumor necrosis factor alpha (TNFα) is attractive, which exerts antitumor effects of immunotherapeutic response and apoptotic/proinflammatory pathways. Nevertheless, the challenges remain how to implement targeted delivery of TNFα to reduce toxicity and manifest the involved signaling mechanism that subdues MDR. Methods: We synthesized a multifunctional nanosytem, in which TNFα covalently bound to doxorubicin (Dox)-loaded pH-responsive mesoporous silica nanoparticles (MSN) through bi-functional polyethylene glycol (TNFα-PEG-MSN-Hydrazone-Dox) as a robust design to overcome MDR. Results: The salient features of this nanoplatform are: 1) by judicious tailoring of TNFα concentration conjugated on MSN, we observed it could lead to a contrary effect of either proliferation or suppression of tumor growth; 2) the MSN-TNFα at higher concentration serves multiple functions, besides tumor targeting and inducer of apoptosis through extrinsic pathway, it inhibits the expression level of p-glycoprotein (P-gp), a cell membrane protein that functions as a drug efflux pump; 3) the enormous surface area of MSN provides for TNFα functionalization, and the nanochannels accommodate chemotherapeutics, Dox; 4) targeted intracellular release of Dox through the pH-dependent cleavage of hydrazone bonds induces apoptosis by the specific intrinsic pathway; and 5) TNFα-PEG-MSN-Hydrazone-Dox (MSN-Dox-TNFα) could infiltrate deep into the 3D spheroid tumor model through disintegration of tight junction proteins. When administered intratumorally in a Dox-resistant mouse tumor model, MSN-Dox-TNFα exhibited a synergistic therapeutic effect through the collective performances of TNFα and Dox. Conclusion: We hereby develop and demonstrate a multifunctional MSN-Dox-TNFα system with concentration-tailored TNFα that can abrogate the drug resistance mechanism, and significantly inhibit the tumor growth through both intrinsic and extrinsic apoptosis pathways, thus making it a highly potential nanomedicine translated in the treatment of MDR tumors.

Rationally Designed Enzyme-Resistant Peptidic Assemblies for Plasma Membrane Targeting in Cancer Treatment.

Peptides are being increasingly important for subcellular targeted cancer treatment to improve specificity and reverse multidrug resistance. However, there has been yet any report on targeting plasma membrane (PM) through self-assembling peptides. A simple synthetic peptidic molecule (tF4) is developed. It is revealed that tF4 is carboxyl esterase-resistant and self-assembles into vesical nanostructures. Importantly, tF4 assemblies interact with PM through orthogonal hydrogen bonding and hydrophobic interaction to regulate cancer cellular functions. Mechanistically, tF4 assemblies induce stress fiber formation, cytoskeleton reconstruction, and death receptor 4/5 (DR4/5) expression in cancer cells. DR4/5 triggers extrinsic caspase-8 signaling cascade, resulting in cell death. The results provide a new strategy for developing enzyme-resistant and PM-targeting peptidic molecules against cancer.

Molecular dissection of anti-colon cancer activity of NARI-29: special focus on H2O2 modulated NF-κB and death receptor signaling.

Accumulating evidence attributes the role of aldose reductase (AR) in modulating ROS and inflammation which are the main factor responsible for cancer progression and drug resistance. Epalrestat is the only AR inhibitor being used in Asian countries. It did not make it to the markets of the USA and Europe due to marginal efficacy as an antioxidant and anti-inflammatory agent owing to difficulty reaching intracellular targets. In our previous studies, we attempted to synthesize the epalrestat analogs and reported that the compound 4-((Z)-5-((Z)-2-Cyano-3-phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) benzoic acid named as NARI-29 has potent AR inhibition compared to epalrestat. In the current study, we aimed to find the effect of NARI-29 on ROS-induced cancer progression and TRAIL resistance in colon cancer in vitro models. In the first part of the study, we demonstrated that the NARI-29 has specific AKR1B1 inhibition and superior drug-like properties than epalrestat using bioinformatics tools. In the second part of the study, it was proven that NARI-29 has induced the hydrogen peroxide-triggered TRAIL-induced apoptosis in the colon cancer cells via modulating the AKR1B1/4HNE/FOXO3a/DR axis. The selective cytotoxicity of NARI-29 (10-fold) compared to epalrestat (4-fold) toward cancer cells is due to its differential ROS regulation and anti-inflammatory activities. Altogether, these data show that NARI-29 may be a potential candidate for AR inhibitors, which will be used to prevent colon cancer progression and as adjuvant therapy for preventing TRAIL resistance.

AKR1B1 is over-expressed in advanced-stage human colon cancer tissuesAKR1B1 mediates resistance to H₂O₂ and TRAIL in human CRC cell linesA co-activation loop exists between NF-κB and AKR1B1 in CRC cell lines to counteract ROSEstablishing epalrestat analog, NARI-29 (4-((Z)-5-((Z)-2-Cyano-3-phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) benzoic acid) as potent anti-colon cancer agentsNARI-29 induced selective apoptosis in colon cancer cells by differentially modulating the ROS and sensitizing TRAIL.

Pivotal role of AKR1B1 in pathogenesis of colitis associated colorectal carcinogenesis.

Identifying the target linking inflammation and oxidative stress to aggravate the disease progression will help to prevent colitis associated carcinogenesis. Since AKR1B1 overexpression is observed in inflammatory diseases and various cancers, we have investigated the role of AKR1B1 in colitis-associated colon carcinogenesis with the aid of epalrestat and its potent analogue NARI-29 (investigational molecule) as pharmacological probes. A TNF-α inducible NF-κB reporter cell line (GloResponse™ NF-κB-RE-luc2P HEK293) and dextran sodium sulfate (DSS) and 1,2 dimethyl hydrazine (DMH))-induced mouse model was used to investigate our hypothesis in vitro and in vivo. Clinically, an increased expression of AKR1B1 was observed in patients with ulcerative colitis. Our in vitro and in vivo findings suggest that the AKR1B1 modulated inflammation and ROS generation for the progression of colitis to colon cancer. AKR1B1 overexpression was observed in DSS + DMH-treated mice colons. Moreover, we could observe histopathological changes like immune cell infiltration, aberrant crypt foci, and tumour formation in DC groups. Mechanistically, we have witnessed modulation of the IKK/IκB/NF-κB and Akt/FOXO-3a/DR axis, increased inflammatory cytokines, increased expression of proliferative markers, Ki-67 and PCNA, and accumulation of ß-catenin in the colon epithelium. However, pharmacological inhibition of AKR1B1 using NARI-29 or EPS has reversed the clinical, histopathological, and molecular alterations induced by DSS + DMH, confirming the obvious role of AKR1B1 in the promotion of colitis-associated carcinogenesis. In conclusion, our findings suggest that AKR1B1 targeted therapy could be a promising strategy for preventing CA-CRC and NARI-29 could be developed as a potent AKR1B1 inhibitor.

Cytochalasin B-Induced Membrane Vesicles from TRAIL-Overexpressing Mesenchymal Stem Cells Induce Extrinsic Pathway of Apoptosis in Breast Cancer Mouse Model.

Tumor-necrosis-factor-associated apoptosis-inducing ligand (TRAIL) is one of the most promising therapeutic cytokines that selectively induce apoptosis in tumor cells. It is known that membrane vesicles (MVs) can carry the surface markers of parental cells. Therefore, MVs are of interest as a tool for cell-free cancer therapy. In this study, membrane vesicles were isolated from TRAIL-overexpressing mesenchymal stem cells using cytochalasin B treatment (CIMVs). To evaluate the antitumor effect of CIMVs-TRAIL in vivo, a breast cancer mouse model was produced. The animals were intratumorally injected with 50 µg of native CIMVs or CIMVs-TRAIL for 12 days with an interval of two days. Then, tumor growth rate, tumor necrotic area, the expression of the apoptosis-related genes CASP8, BCL-2, and BAX and the level of CASP8 protein were analyzed. A 1.8-fold increase in the CAS8 gene mRNA and a 1.7-fold increase in the CASP8 protein level were observed in the tumors injected with CIMVs-TRAIL. The expression of the anti-apoptotic BCL-2 gene in the CIMV-TRAIL group remained unchanged, while the mRNA level of the pro-apoptotic BAX gene was increased by 1.4 times, which indicated apoptosis activation in the tumor tissue. Thus, CIMVs-TRAIL were able to activate the extrinsic apoptosis pathway and induce tumor cell death in the breast cancer mouse model.

Extrinsic apoptosis and senescence involved in growth kinetics of seminoma to cisplatin.

Seminoma is the most common type of testicular germ cell tumour and is highly sensitive to cisplatin therapy, which has not been fully elucidated. In this study, we comprehensively monitored dynamic changes of TCam-2 cells after cisplatin treatment. At an early stage, we found that both low and high concentrations of cisplatin induced the S-phase arrest in TCam-2 cells. By contrast, the G0G1 arrest was caused by cisplatin in teratoma NTERA-2 cells. Afterwards, high concentrations of cisplatin promoted the extrinsic apoptosis and high expressions of related genes (Fas/FasL-caspase-8/-3) in TCam-2 cells. However, when decreasing the cisplatin, the apoptotic cells were significantly reduced, and accompanied by cells showing senescence-like morphology, positive SA-ß-gal staining and up-regulation of senescence-related genes (p53, p21 and p16). Furthermore, the cell cycle analysis revealed that most of the senescent TCam-2 cells were irreversibly arrested in the G2M phase. G2M arrest was also observed in NTERA-2 cells treated with a low concentration of cisplatin, while no senescence-related phenotype was discovered. In addition, we detected the γ-H2AX, a DNA damage marker protein, and reactive oxygen species (ROS) and found both of which were elevated with the increase of cisplatin in TCam-2 cells. In conclusion, the extrinsic apoptosis and senescence are involved in the growth kinetics of TCam-2 cells to cisplatin, which provide some implications for studies on cisplatin and seminoma.

Brevilin A Isolated from Centipeda minima Induces Apoptosis in Human Gastric Cancer Cells via an Extrinsic Apoptotic Signaling Pathway.

Brevilin A, which has anticancer activities against a range of cancers, is an abundant constituent of the medicinal herb Centipeda minima (L.) A. Braun & Asch, which has also been reported to have anticancer activity against breast cancer cells. However, the anticancer activities of C. minima and brevilin A against human gastric cancer have yet to be reported. In this study, we aimed to evaluate the cytotoxicity and molecular basis underlying the anticancer activities of extracts of C. minima (CMX) and brevilin A against human gastric cancer (AGS) cells. We deduced the potential targets and mechanisms underlying the anticancer activity of brevilin A based on a network pharmacology approach. CCND1, CDK4, and BCL2L1 were identified as the key anticancer genes targeted by brevilin A. Cytotoxicity analyses revealed that CMX and brevilin A reduced the viability of AGS cells to levels below 50% (9.73 ± 1.29 µg/mL and 54.69 ± 1.38 µM, respectively). Furthermore, Hoechst 33342, annexin V, and propidium iodide staining and western blot analyses revealed that CMX and brevilin A promoted a significant induction of apoptotic cell death by upregulating the expression of cleaved caspase-8 and cleaved caspase-3 and reducing the ratio of Bax to Bcl-2, which is partially consistent with the findings of our network pharmacology analysis. Collectively, our observations indicate that CMX and brevilin A are novel sources of herbal medicine with potential utility as effective agents for the treatment of gastric cancer.

Celastrol Inhibited Human Esophageal Cancer by Activating DR5-Dependent Extrinsic and Noxa/Bim-Dependent Intrinsic Apoptosis.

Esophageal squamous cell carcinoma (ESCC) is one of the deadliest digestive system cancers worldwide lacking effective therapeutic strategies. Recently, it has been found that the natural product celastrol plays an anti-cancer role in several human cancers by inducing cell cycle arrest and apoptosis. However, it remains elusive whether and how celastrol suppresses tumor growth of ESCC. In the present study, for the first time, we demonstrated that celastrol triggered both extrinsic and intrinsic apoptosis pathways to diminish the tumor growth of ESCC in vivo and in vitro. Mechanistic studies revealed that celastrol coordinatively induced DR5-dependent extrinsic apoptosis and Noxa-dependent intrinsic apoptosis through transcriptional activation of ATF4 in ESCC cells. Furthermore, we found that the FoxO3a-Bim pathway was involved in the intrinsic apoptosis of ESCC cells induced by celastrol. Our study elucidated the tumor-suppressive efficacy of celastrol on ESCC and revealed a previously unknown mechanism underlying celastrol-induced apoptosis, highlighting celastrol as a promising apoptosis-inducing therapeutic strategy for ESCC.

Sialic acids on B cells are crucial for their survival and provide protection against apoptosis.

Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas-deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.

Targeting Apoptosis in Cancer.

PURPOSE OF REVIEW: Apoptosis is a major mechanism of cancer cell death. Thus, evasion of apoptosis results in therapy resistance. Here, we review apoptosis modulators in cancer and their recent developments, including MDM2 inhibitors and kinase inhibitors that can induce effective apoptosis. RECENT FINDINGS: Both extrinsic pathways (external stimuli through cell surface death receptor) and intrinsic pathways (mitochondrial-mediated regulation upon genotoxic stress) regulate the complex process of apoptosis through orchestration of various proteins such as members of the BCL-2 family. Dysregulation within these complex steps can result in evasion of apoptosis. However, via the combined evolution of medicinal chemistry and molecular biology, omics assays have led to innovative inducers of apoptosis and inhibitors of anti-apoptotic regulators. Many of these agents are now being tested in cancer patients in early-phase trials. We believe that despite a sluggish speed of development, apoptosis targeting holds promise as a relevant strategy in cancer therapeutics.

Multiple Autonomous Cell Death Suppression Strategies Ensure Cytomegalovirus Fitness.

Programmed cell death pathways eliminate infected cells and regulate infection-associated inflammation during pathogen invasion. Cytomegaloviruses encode several distinct suppressors that block intrinsic apoptosis, extrinsic apoptosis, and necroptosis, pathways that impact pathogenesis of this ubiquitous herpesvirus. Here, we expanded the understanding of three cell autonomous suppression mechanisms on which murine cytomegalovirus relies: (i) M38.5-encoded viral mitochondrial inhibitor of apoptosis (vMIA), a BAX suppressor that functions in concert with M41.1-encoded viral inhibitor of BAK oligomerization (vIBO), (ii) M36-encoded viral inhibitor of caspase-8 activation (vICA), and (iii) M45-encoded viral inhibitor of RIP/RHIM activation (vIRA). Following infection of bone marrow-derived macrophages, the virus initially deflected receptor-interacting protein kinase (RIPK)3-dependent necroptosis, the most potent of the three cell death pathways. This process remained independent of caspase-8, although suppression of this apoptotic protease enhances necroptosis in most cell types. Second, the virus deflected TNF-mediated extrinsic apoptosis, a pathway dependent on autocrine TNF production by macrophages that proceeds independently of mitochondrial death machinery or RIPK3. Third, cytomegalovirus deflected BCL-2 family protein-dependent mitochondrial cell death through combined TNF-dependent and -independent signaling even in the absence of RIPK1, RIPK3, and caspase-8. Furthermore, each of these cell death pathways dictated a distinct pattern of cytokine and chemokine activation. Therefore, cytomegalovirus employs sequential, non-redundant suppression strategies to specifically modulate the timing and execution of necroptosis, extrinsic apoptosis, and intrinsic apoptosis within infected cells to orchestrate virus control and infection-dependent inflammation. Virus-encoded death suppressors together hold control over an intricate network that upends host defense and supports pathogenesis in the intact mammalian host.

Fangchinoline Inhibits Human Esophageal Cancer by Transactivating ATF4 to Trigger Both Noxa-Dependent Intrinsic and DR5-Dependent Extrinsic Apoptosis.

Esophageal squamous cell carcinoma (ESCC) is a recalcitrant cancer. The Chinese herbal monomer fangchinoline (FCL) has been reported to have anti-tumor activity in several human cancer cell types. However, the therapeutic efficacy and underlying mechanism on ESCC remain to be elucidated. In the present study, for the first time, we demonstrated that FCL significantly suppressed the growth of ESCC both in vitro and in vivo. Mechanistic studies revealed that FCL-induced G1 phase cell-cycle arrest in ESCC which is dependent on p21 and p27. Moreover, we found that FCL coordinatively triggered Noxa-dependent intrinsic apoptosis and DR5-dependent extrinsic apoptosis by transactivating ATF4, which is a novel mechanism. Our findings elucidated the tumor-suppressive efficacy and mechanisms of FCL and demonstrated FCL is a potential anti-ESCC agent.

Peptide amphiphile nanofiber hydrogel delivery of Sonic hedgehog protein to the penis and cavernous nerve suppresses intrinsic and extrinsic apoptotic signaling mechanisms, which are an underlying cause of erectile dysfunction.

Erectile dysfunction (ED) is a common and debilitating condition with high impact on quality of life. An underlying cause of ED is apoptosis of penile smooth muscle, which occurs with cavernous nerve injury, in prostatectomy, diabetic and aging patients. We are developing peptide amphiphile (PA) nanofiber hydrogels as an in vivo delivery vehicle for Sonic hedgehog protein to the penis and cavernous nerve to prevent the apoptotic response. We examine two important aspects required for clinical application of the biomaterials: if SHH PA suppresses intrinsic (caspase 9) and extrinsic (caspase 8) apoptotic mechanisms, and if suppressing one apoptotic mechanism forces apoptosis to occur via a different mechanism. We show that SHH PA suppresses both caspase 9 and 8 apoptotic mechanisms, and suppressing caspase 9 did not shift signaling to caspase 8. SHH PA has significant clinical potential as a preventative ED therapy, by management of intrinsic and extrinsic apoptotic mechanisms.

Iridin Induces G2/M Phase Cell Cycle Arrest and Extrinsic Apoptotic Cell Death through PI3K/AKT Signaling Pathway in AGS Gastric Cancer Cells.

Iridin is a natural flavonoid found in Belamcanda chinensis documented for its broad spectrum of biological activities like antioxidant, antitumor, and antiproliferative effects. In the present study, we have investigated the antitumor potential of iridin in AGS gastric cancer cells. Iridin treatment decreases AGS cell growth and promotes G2/M phase cell cycle arrest by attenuating the expression of Cdc25C, CDK1, and Cyclin B1 proteins. Iridin-treatment also triggered apoptotic cell death in AGS cells, which was verified by cleaved Caspase-3 (Cl- Caspase-3) and poly ADP-ribose polymerase (PARP) protein expression. Further apoptotic cell death was confirmed by increased apoptotic cell death fraction shown in allophycocyanin (APC)/Annexin V and propidium iodide staining. Iridin also increased the expression of extrinsic apoptotic pathway proteins like Fas, FasL, and cleaved Caspase-8 in AGS cells. On the contrary, iridin-treated AGS cells did not show variations in proteins related to an intrinsic apoptotic pathway such as Bax and Bcl-xL. Besides, Iridin showed inhibition of PI3K/AKT signaling pathways by downregulation of (p-PI3K, p-AKT) proteins in AGS cells. In conclusion, these data suggest that iridin has anticancer potential by inhibiting PI3K/AKT pathway. It could be a basis for further drug design in gastric cancer treatment.

Anti-proliferation and apoptosis-inducing effects of sodium aescinate on retinoblastoma Y79 cells.

AIM: To investigate the anti-proliferation and apoptosis-inducing effects of sodium aescinate (SA) on retinoblastoma Y79 cells and its mechanism. METHODS: Y79 cells were cultured at different drug concentrations for different periods of time (24, 48, and 72h). The inhibitory effect of SA on proliferation of Y79 cells was detected by the cell counting kit-8 (CCK-8) assay, and the morphology of Y79 cells in each group was observed under an inverted microscope. An IC50 of 48h was selected for subsequent experiments. After pretreatment with SA for 24 and 48h, cellular DNA distribution and apoptosis were detected by flow cytometry. Real-time qunatitative polymerase chain reaction (RT-qPCR) and Western blot were used to assess changes in related genes (CDK1, CyclinB1, Bax, Bcl-2, caspase-9, caspase-8, and caspase-3). RESULTS: SA inhibited proliferation and induced apoptosis of Y79 cells in a time-dependent and concentration-dependent manner. Following its intervention in the cell cycle pathway, SA can inhibit the expression of CDK1 and CyclinB1 at the mRNA and protein levels, and block cells in the G2/M phase. In caspase-related apoptotic pathways, up-regulation of Bax and down-regulation of Bcl-2 caused caspase-9 to self-cleave and further activate caspase-3. What's more, the caspase-8-mediated extrinsic apoptosis pathway was activated, and the activated caspase-8 was released into the cytoplasm to activate caspase-3, which as a member of the downstream apoptotic effect group, initiates a caspase-cascade reaction that induces cell apoptosis. CONCLUSION: SA inhibits the proliferation of Y79 cells by arresting the cell cycle at the G2/M phase, and induces apoptosis via the caspase-related apoptosis pathway, indicating that SA may have promising potential as a chemotherapeutic drug.

TNF Signaling Dictates Myeloid and Non-Myeloid Cell Crosstalk to Execute MCMV-Induced Extrinsic Apoptosis.

Cytomegaloviruses all encode the viral inhibitor of caspase-8-induced apoptosis (vICA). After binding to this initiator caspase, vICA blocks caspase-8 proteolytic activity and ability to activate caspase-3 and/or caspase-7. In this manner, vICA has long been known to prevent apoptosis triggered via tumor necrosis factor (TNF) family death receptor-dependent extrinsic signaling. Here, we employ fully wild-type murine cytomegalovirus (MCMV) and vICA-deficient MCMV (∆M36) to investigate the contribution of TNF signaling to apoptosis during infection of different cell types. ∆M36 shows the expected ability to kill mouse splenic hematopoietic cells, bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC). Antibody blockade or genetic elimination of TNF protects myeloid cells from death, and caspase-8 activation accompanies cell death. Interferons, necroptosis, and pyroptotic gasdermin D (GSDMD) do not contribute to myeloid cell death. Human and murine fibroblasts or murine endothelial cells (SVEC4-10) normally insensitive to TNF become sensitized to ∆M36-induced apoptosis when treated with TNF or TNF-containing BMDM-conditioned medium. We demonstrate that myeloid cells are the natural source of TNF that triggers apoptosis in either myeloid (autocrine) or non-myeloid cells (paracrine) during ∆M36 infection of mice. Caspase-8 suppression by vICA emerges as key to subverting innate immune elimination of a wide variety of infected cell types.