Extrinsic Apoptosis Pathway Research Articles

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.

In vitro chondrotoxicity of bupivacaine, levobupivacaine and ropivacaine and their effects on caspase activity in cultured canine articular chondrocytes.

Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.

Comparative studies on in vitro antitumor activities and apoptosis-inducing effects of enantiomeric ruthenium(II) complexes.

On the basis of our previous comparative studies on the DNA binding of a pair of ruthenium(II) complex enantiomers, Δ-[Ru(bpy)2PBIP]2+ and Λ-[Ru(bpy)2PBIP]2+ {bpy = 2,2'-bipyridine, PBIP = 2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline}, in this study, their antitumor activities and mechanisms were further investigated comparatively. The cytotoxicity assay demonstrated that both the enantiomers exerted selective antiproliferative effects on cancer cell lines A2780 and PC3. Fluorescence localization experiments suggested that both the enantiomers effectively permeated the nucleus of HeLa cells and co-localized with DNA, resulting in their DNA damage and apoptosis. Flow cytometry experiments showed that the apoptosis was enhanced by increasing the concentration of each enantiomer. Western blotting analyses indicated that both extrinsic and intrinsic apoptosis pathways were activated by the two enantiomers. miRNA microarray analyses displayed that both the enantiomers up- and downregulated multiple miRNAs, some of which were predicted to be associated with carcinogenesis. The above experimental results also showed that the Δ-enantiomer exerted a more potent antitumor activity, a higher efficiency of entering cancer cells and a stronger apoptosis-inducing effect compared with the Λ-enantiomer. Combined with the previously published research results, experimental results from this study implied that the antitumor activity of a metal complex might have originated from the conformation change of DNA in tumor cells caused by the intercalation of the complex, that the antitumor mechanism of a metal complex could be related to its DNA-binding mode, and that the antitumor efficiency of a metal complex could result from its DNA-binding strength.

Ethylcoprostanol modulates colorectal cancer cell proliferation and mitigates cytotoxicity of cholesterol metabolites in non-tumor colon cells.

Sterols can be metabolized by gut microbiota. The cholesterol metabolites have been proposed as promoters of colorectal cancer (CRC), while the effect of plant sterol metabolites is unknown. This study aimed to evaluate the cytotoxicity of metabolites from cholesterol (coprostanol, cholestanol, coprostanone and cholestenone) and β-sitosterol (ethylcoprostanol) on human colon tumor (Caco-2) and non-tumor (CCD-18Co) cells at physiological concentrations (9-300 μM) and exposure time (24 h). Ethylcoprostanol reduced the tumor cell proliferation (MTT), showing in flow cytometry assays induction of apoptosis via production of reactive oxygen species (ROS) and ceramide. Transcriptomic analysis (qPCR) showed activation of the intrinsic apoptosis pathway (BAX/BCL2 ratio and CASP9 increased), accompanied by downregulation of the p21 gene. Cholesterol metabolites, mainly the most hydrophobic, induced apoptosis and G0/G1 phase arrest in non-tumor cells through overproduction of ROS. Both the intrinsic and extrinsic (CASP8 increased) apoptosis pathways occurred. In turn, a reduction in the expression of the cyclin E1 gene confirmed the cell cycle arrest. In addition, ethylcoprostanol protected non-tumor cells from the most cytotoxic cholesterol metabolite (cholestenone). In conclusion, ethylcoprostanol is a promising candidate as a therapeutic adjuvant in CRC, while cholesterol metabolites could act as CRC promoters through their cytotoxicity.

FoxM1 knockdown enhanced radiosensitivity of esophageal cancer by inducing apoptosis.

Radioresistance is a main reason for local recurrence of esophageal squamous cell carcinoma (ESCC). Forkhead box M1 (FoxM1) is implicated in cancer progression and chemoresistance. This study aims to determine the role of FoxM1 in ESCC radioresistance. We found that FoxM1 protein was upregulated in ESCC tissues compared with adjacent normal tissues. In vitro assays revealed that following irradiation, Eca-109, TE-13, and KYSE-150 cells had increased levels of FoxM1 protein. FoxM1 knockdown resulted in significantly reduced colony formation and increased cell apoptosis following irradiation. Moreover, FoxM1 knockdown induced ESCC cells to accumulate in the radiosensitive G2 /M phase and impeded the repair of radiation-induced DNA damage. Mechanistic studies indicated that radiosensitization of ESCC enhanced by FoxM1 knockdown was associated with increased BAX/BCL2 ratio as well as downregulated Survivin and XIAP, followed by the activation of both extrinsic and intrinsic apoptosis pathways. In xenograft mouse model, the combination of radiation and FoxM1-shRNA led to a synergistic anti-tumor effect. In conclusion, FoxM1 is a promising target to enhance radiosensitivity of ESCC.

Combinational Therapy of Acute Lymphoblastic Leukemia with Cyclophosphamide and BV6 Synergistically Induces Apoptosis in Leukemic Cells

Background: Acute lymphoblastic leukemia (ALL), a hematological malignancy, is associated with the accumulation of lymphoblasts in hematopoietic tissues and the peripheral blood (PB). A major obstacle in ALL treatment is the failure to undergo apoptosis, thus resulting in relapses. Cyclophosphamide is an alkylating agent widely used to treat various cancers. BV6 is another agent, under preclinical and clinical trials, which inhibits the function of inhibitors of apoptosis proteins (IAPs) in cells. According to previous studies, IAPs are highly overexpressed in ALL cells. Also, the intrinsic apoptosis pathway is reported to be dysfunctional in ALL cells, making BV6 a new promising option due to its potential to induce extrinsic cell death as well. Methods: In the current study, the inhibitory effect of the combinational treatment of cyclophosphamide and BV6 was studied on the cell growth of eleven ALL patients using an MTT test. Subsequently, the effect of the treatment was studied on TNF-α secretion as well. Finally, we measured the DNA fragmentation rates of treated cells as well. Results: The results indicated a synergistic effect between the drugs on the viability of cells. Also, the impact of the cotreatment was increased in a time-dependent manner. We also found that BV6, but not cyclophosphamide, induces TNF-α secretion, activating the extrinsic apoptosis pathway as well. Conclusion: BV6, by inducing intrinsic and extrinsic pathways, overcomes cyclophosphamide resistance. These results suggest the combinational use of BV6 and cyclophosphamide for the treatment of ALL patients. Inhibition of IAPs helps overcome the failure to undergo apoptosis and can improve the prognosis of ALL patients.

Synthesize of Bi2O3/Gln-TSC nanoparticles and evaluation of their toxicity on prostate cancer cells and expression of CASP8, BAX, and Bcl-2 genes.

Due to the high prevalence and considerable increase of prostate cancer, finding novel therapeutic compounds for the treatment of prostatic cancer has been the goal of many researches. In this study, we aimed to fabricate the Bismuth oxide (Bi2O3) NPs, functionalized with glutamine (Gln) and conjugated with Thiosemicarbazide (TSC). Then, the anticancer mechanism of the synthesized NPs was investigated using the cellular and molecular tests including MTT assay, Flow cytometry, Caspase-3 activity assay, Hoechst staining and Real Time PCR. The FT-IR and XRD assays confirmed the identity of the synthesized Bi2O3/Gln-TSC NPs. The size range of the synthesized spherical particles was 10-60nm and the zeta potential was -23.8mV. The purity of the NPs was confirmed by EDX-mapping analysis. The Bi2O3/Gln-TSC was considerably more toxic for prostate cancer cells than normal human cells and the IC50 was calculated 35.4 and 305µg/mL, respectively. The exposure to the NPs significantly increased the frequency of apoptotic cells from 4.7 to 75.3%. Moreover, the expression of the CASP8, BAX, and Bcl-2 genes after exposure to the NPs increased by 2.8, 2.3, and 1.39 folds. Treating the cancer cells with Bi2O3/Gln-TSC increased the activity of the Caspase-3 protein and apoptotic morphological features were observed by Hoechst staining in the treated cells. This work showed that Bi2O3/Gln-TSC has considerable cytotoxicity for prostate cancer cells and could inducing both intrinsic and extrinsic pathways of apoptosis.

Novel benzo chromene derivatives: design, synthesis, molecular docking, cell cycle arrest, and apoptosis induction in human acute myeloid leukemia HL-60 cells

A series of benzo[h]chromenes, benzo[f]chromenes, and benzo[h]chromeno[2,3-d]pyrimidines were prepared. All the newly synthesised compounds were selected by National Cancer Institute for single-dose testing against 60 cell lines. Benzo[h]chromenes 5a and 6a showed promising anti-cancer activity and selected for the five-dose testing. Compounds 5a and 6a suppressed cell growth in HL-60 by the induction of cell cycle arrest, which was confirmed using flow cytometry and Annexin V-FITC/PI assays showed at the G1/S phase by regulating the expression of CDK-2/CyclinD1, triggering cell apoptosis by activating both the extrinsic (Fas/Caspase 8) and intrinsic (Bcl-2/Caspase 3) apoptosis pathways, which were determined by the western blot. Benzo[h]chromenes 5a and 6a decreased the protein expression levels of Bcl-2, CDK-2, and CyclinD1 and increased the expression of caspase 3, caspase 8, and Fas. In silico molecular analysis of compounds 5a and 6a in CDK-2 and Bcl-2 was performed.

P2X7R and TNFR1 antagonism neuroprotects rodent retinal ganglion cells in a sepsis model

AbstractPurpose: i/ To analyse the time course of retinal ganglion cell death (RGCs) after systemic injection of LPS in mice. ii/ To analyse the neuroprotective effect of P2X7 or TNFR1 receptor antagonism after LPS treatment.Methods: Male C57BL/6J mice were given an intraperitoneal injection of increasing doses of LPS and retinas were analysed at 3 days. Once the dose was set, to test the neuroprotective effect of the antagonism of the P2X7 and the TNFR1 receptors, antagonists were administered subcutaneously or intraperitoneally, alone or in combination. Control groups were intact animals and animals treated with LPS + vehicle. In all groups, retinas were dissected flat and the total number of RGCs immunodetected with Brn3a was quantified.Results: Significant death of RGCs was observed with doses higher than 4 mg/kg of LPS. In LPS treated animals, the loss of RGCs was observed at 3 days a progressed up to 7 days when 31% of the original RGC population was lost. From 7 to 21 days RGC loss did not progress. Antagonism of P2X7R or TNFR1 rescued 95% and 92% respectively of the original RGC population. Combination of both treatments did not improve each one alone.Conclusions: A single injection of LPS causes RGC death which is rescued by more than 90% by antagonizing the inflammasome or the extrinsic pathway of apoptosis.

The DLBCL Axis of Low TNFRSF10B Expression, TP53 Loss, and a Cold Immune Microenvironment May be Addressed By the Synergistic Combination of Eprenetapopt and Idasnutlin

Background: Genomically-targeted Diffuse Large B-cell (DLBCL) treatments for refractory or relapsing patients remain a clinical need alongside the growing success of Chimeric Antigen T-cells (CAR-T), which have ushered in durable responses in 40-70% of patients. However, recent studies have identified factors indicative of an inferior CAR-T response. These include a "cold" immune microenvironment, downregulation of extrinsic cell death factors such as TNFRSF10B, and alterations to the TP53 tumor suppressor. Few precision treatments exist capable of addressing the consequences, although two avenues are being pursued in clinical trials for lymphoma: inhibition of the TP53 negative regulator MDM2 via Idasnutlin and TP53 reactivation via the unique molecule Eprenetapopt (APR-246). Herein, we report the first observations of synergy between Idasnutlin and Eprenetapopt within DLBCL cell lines and data supporting that TP53 restoration may revive extrinsic apoptosis and immunological pathways capable of modulating CAR-T success. Methods: We analyzed 2 de-novo DLBCL patient cohorts (Xu-Monette et. al. 2020 and the TCGA) for differential expression and pathway analysis between TP53-mutant and Low TNFRSF10B (-1 Standard Deviation) patients versus normal counterparts. We integrated data from 3 additional sources to uncover actionable vulnerabilities within patients faced with dismal CAR-T response: differential gene expression between TP53-mutant and WT rrDLBCL patients (Shouval et. al. 2022), in-vitro CAR-T sgRNA-cytotoxicity scores (Dufva et. al. 2020), and gene expression following MDM2 inhibition (Louise et. al. 2021). We next assayed Idasnutlin and Eprenetapopt vs. 7 DLBCL cell lines, 5 of which harbor TP53 alterations, across a series of 9 concentrations (78nM-20µM) across 3-9 replicates. We followed these results by combining treatments in the TP53-impaired DHL6 and U2932 cell lines, analyzing results using the HSA synergy model. Lastly, we performed Western blot analysis to measure protein expression of TNFRSF10B following 24-hour single-agent or combination treatment. Results: Integrative analyses support that DNA alterations to TP53 are associated with significantly reduced T-cell activation and extrinsic apoptosis genes. TNFSRF10B was significantly reduced in these cases as well (FDR = 0.0034), and genes with significantly reduced expression in both TP53-mut and Low TNFRSF10B expression were associated with the loss of Immune System Development genes (FDR = 3.66E-09). The Staudt lab IFNG-Up and STAT-Up gene signature pathways were significantly reduced in TP53-mut patients (P < 0.0001, P = 0.483) and in Low TNFRSF10B Patients (P < 0.0001, P = 0.488). TNFRSF10B was also the only gene with significantly reduced expression in TP53-mut rrDLBCL patients, importance for CAR-T cytotoxicity, and increased expression following MDM2 inhibition. Our Western blot results support these observations, showcasing upregulation of TNFRS10B following treatment. Idasnutlin and Eprenetapopt assays vs. DLBCL cell lines demonstrated dose and TP53-dependent cell inhibition, with ED50 values ranging between 880nM to 19.6µM, independent of COO. The homozygous-impaired TP53 cell line Karpas-422 was the least sensitive. The molecules were next combined vs. DHL6 and U2932 cell lines, resulting in synergistic reduction of cell viability and HSA synergy scores of 10.94, and 5.32, respectively. Combined treatments of 400 nM Idasnutlin and Eprenetapopt reduced cell viability significantly greater than single agent treatments at the same concentration in DHL6 (P = 0.0001; P = 0.0176) and U2932 (P = 0.0012; P = 0.0322). Conclusions: Our results suggest the presence of an interconnected genomic DLBCL phenotype characterized by TP53 loss, reduced TNFRSF10B expression, and a debilitated immune presence, collectively predictive of an inferior CD19 CAR-T response. We applied the targeted therapies Eprenetapopt and Idasnutlin vs. cell line models, with both displaying anti-tumor activity as single agents. Most importantly, we observed synergistic activity in TP53-imparied DLBCL cell lines when the molecules were combined. These results are buoyed by integrative and Western blot analyses that highlight the promise of TNFRSF10B revival afforded by these agents, revealing a potential avenue to address inferior CAR-T response rates faced by patients harboring TP53 alterations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

DDDR-20. TARGETING THE SELECTIVE VULNERABILITY OF IDH-MUTANT GLIOMA WITH ZOTIRACICLIB

Abstract BACKGROUND IDH mutations are common in diffuse gliomas and confer an increased dependency on oxidative mitochondrial function. Zotiraciclib, a multi-kinase inhibitor, not only suppresses CDK9-mediated gene transcription process but also causes mitochondrial dysfunction in preclinical models. We investigated the selective activity of zotiraciclib in IDH-mutant gliomas. METHODS Cytotoxicity of zotiraciclib was determined in patient-derived glioma cells with/without IDH mutations. Immunoblotting and flow cytometry were used to evaluate the cell death pathways. Real-time ATP production was measured by Seahorse assay. RESULTS The EC50 of zotiraciclib in IDH-wt cells was twice as high as in IDH-mutant cells. In low-dose zotiraciclib (15nM)-treated IDH-mutant cells, RNA Pol II phosphorylation and expression of antiapoptotic proteins, including XIAP, MCL-1 and survivin were decreased significantly. Cleaved caspase 3/7, 8, 9 and LC3B were also detected in treated IDH-mutant cells but not in IDH-wt cells, suggesting activation of intrinsic and extrinsic cell apoptosis pathways, and subsequent autophagy of IDH-mutant cells. Low-dose treated IDH-mutant, but not IDH-wt cells, induced decreased expression of a panel of mitochondrial respiration genes, and mitochondrial respiration complexes, cellular ATP depletion and increased ROS production, suggesting a greater disruption of mitochondrial function in zotiraciclib-treated IDH-mutant cells. Zotiraciclib treatment prolonged the survival in mice bearing intracranial IDH-mutant gliomas (30 days in DMSO-treated group vs 33 days in zotiraciclib-treated group, p = 0.01), but not in mice with glioma established with the isogenic IDH-wt glioma cells (18 days in DMSO-treated vs 20 days in zotiraciclib-treated group, p = 0.10). CONCLUSIONS There is selective activity of zotiraciclib in IDH-mutated glioma. Low-dose exposure of zotiraciclib induces cell death and suppresses transcriptional process and mitochondrial biogenesis in IDH-mutant glioma, not in IDH-wt tumors. These findings support a clinical trial testing zotiraciclib in IDH-mutated glioma. The treatment efficacy seen even at low concentrations further supports testing it as a single agent, thereby reducing toxicity concerns.

Ketoprofen suppresses triple negative breast cancer cell growth by inducing apoptosis and inhibiting autophagy.

Triple-negative breast cancer (TNBC) is an invasive phenotype with undesirable clinical features, poor prognosis, and therapy resistance. Ketoprofen is a Non-steroidal anti-inflammatory drug (NSAID) with anti-tumor properties. To investigate the effects of Ketoprofen on apoptosis and autophagy in TNBC cell line MDA-MB-231. The cytotoxic activity of Ketoprofen was assayed by the MTS method. Flowcytometry was utilized to measure the number of apoptotic MDA-MB-231 cells. The expression levels of apoptosis and autophagy markers, JAK2 and STAT3 were determined using quantitative real time-PCR (qRT-PCR) and western blotting methods. Ketoprofen significantly decreased the proliferation of MDA-MB-231 cells compared to control cells. It also considerably induced apoptosis and apoptotic markers in these cells in comparison to controls. Treating the MADA-MB-231 cell line with Ketoprofen had an inhibitory effect on autophagy markers in this cell line. The use of FasL, as a death ligand, and ZB4, as an antibody that blocks the extrinsic pathway of apoptosis, revealed the involvement of the extrinsic pathway in the apoptosis-stimulating effect of Ketoprofen in the MADA-MB-231 cell line. Ketoprofen also hindered the phosphorylation and activation of JAK2 and STAT molecules leading to the inhibition of the JAK/STAT pathway in this TNBC cell line. The outcomes of this study uncovered the anti-TNBC activity of Ketoprofen by inducing apoptosis and inhibiting viability and autophagy in MADA-MB-231 cells. Our data also suggested that Ketoprofen impedes apoptosis in TNBC cells by two different mechanisms including the induction of the extrinsic apoptotic pathway and inhibition of the JAK/STAT signaling.

Iron oxide-alginate-berbamine nanocomposites trigger reactive oxygen species-induced apoptosis through down-regulation of PI3K/AKT signaling in human bladder cancer cells

Globally, bladder cancers are the most common cancers with 81,180 new cases in 2022. Being one of the most important urological cancers it has 17,100 estimated deaths in 2022. Unequivocally the best-known treatment so far is radical cystectomy followed by chemotherapy. Despite the treatment strategies including surgical intervention and adjuvant therapy recurrent rate and tumor development prevention remains as a potential problem. Hence targeted treatment strategies gained more interests and the current exploration examines the potential of iron oxide-alginate-berbamine nanocomposites on human bladder cancer T24 cells. The synthesis of iron oxide-alginate-berbamine nanocomposites was with iron (III) nitrate hexahydrate, sodium alginate, and the phytocomponent berbamine. The iron oxide-alginate-berbamine nanocomposites were then treated with human bladder cancer T24 cells. Interestingly, there was a significant dose-dependent reduction in the cell survival rate (2.5–15 µg/ml). Furthermore, the nanocomposites activated caspase-3,8 and -9 almost two-fold. These caspases are the key players in extrinsic and intrinsic apoptosis pathways. The nanocomposites also elevated the ROS status, thereby inactivating the PI3K/Akt signaling pathway. Thus, the current outcomes indicate that iron oxide-alginate-berbamine nanocomposites could effectively inhibit proliferation and induce apoptosis and block the PI3K/Akt pathway in T24 cells.

Dual inhibition of BET and HAT/p300 suppresses colorectal cancer via DR5- and p53/PUMA-mediated cell death.

BackgroundColorectal cancer (CRC) frequently has a dysregulated epigenome causing aberrant up-regulation of oncogenes such as c-MYC. Bromodomain and extra-terminal domain (BET) proteins and histone acetyltransferases (HAT) are epigenetic regulatory proteins that create and maintain epigenetic states supporting oncogenesis. BET inhibitors and HAT inhibitors are currently being investigated as cancer therapeutics due to their ability to suppress cancer-promoting epigenetic modifiers. Due to the extensive molecular crosstalk between BET proteins and HAT proteins, we hypothesized that dual inhibition of BET and HAT could more potently inhibit CRC cells than inhibition of each individual protein.MethodsWe investigated the activity and mechanisms of a dual BET and HAT inhibitor, NEO2734, in CRC cell lines and mouse xenografts. MTS, flow cytometry, and microscopy were used to assess cell viability. qPCR, Western blotting, and immunofluorescent staining were used to assess mechanisms of action.ResultsWe found that NEO2734 more potently suppresses CRC cell growth than first generation BET inhibitors, regardless of the status of common CRC driver mutations. We previously showed that BET inhibitors upregulate DR5 to induce extrinsic apoptosis. In the current study, we show that NEO2734 treatment induces CRC cell apoptosis via both the intrinsic and extrinsic apoptosis pathways. NEO2734 increases p53 expression and subsequently increased expression of the p53-upregulated mediator of apoptosis (PUMA), which is a critical mechanism for activating intrinsic apoptosis. We demonstrate that inhibition of either the intrinsic or extrinsic branches of apoptosis partially rescues CRC cells from NEO2734 treatment, while the dual inhibition of both branches of apoptosis more strongly rescues CRC cells from NEO2734 treatment. Finally, we show that NEO2734 monotherapy is able to suppress tumor growth in CRC xenografts by inducing apoptosis.ConclusionsOur study demonstrates NEO2734 potently suppresses CRC cells in vitro and in vivo by simultaneously upregulating PUMA and DR5 to induce cell death. Further studies of NEO2734 for treating CRC are warranted.

Establishment of the first cell line from the small yellow croaker (Larimichthys polyactis) and its application in unraveling the mechanism of ROS-induced apoptosis under hypoxia

Cell lines play an important role in studying biological processes in vitro. In this study, we aimed to establish and characterize the first cell line isolated from the small yellow croaker (Larimichthys polyactis), an economically important marine fish in China. The small yellow croaker fry (SYCF) cells proliferated well in Leibovitz's 15 medium containing 2% antibiotics, 0.1 M NaCl, and 10% fetal bovine serum at 27 °C. Sequence analyses of mitochondrial genes confirmed the origin of SYCF cells. Furthermore, we unraveled the mechanism of hypoxia-induced apoptosis, a major threat to aquatic animals using the SYCF cell line. The HIF-PHD pathway of the SYCF cells can be activated under hypoxic stress, suggesting that this cell line is impressible for hypoxia and can serve as a superb model in vitro to study the cell physiology in L. polyactis in a hypoxic setting. We found that hypoxia results in morphological abnormalities, excess reactive oxygen species (ROS) production, and dysfunctional redox homeostasis, indicating the occurrence of oxidative stress in the SYCF cells. In addition, decreased cell viability and severe apoptosis were detected under hypoxic conditions. Through monitoring the indicators of the intrinsic and extrinsic apoptosis pathways, including the mitochondrial membrane potential, the expression of bax, bcl2, bnip3, and fas, Bcl2, and Caspase-3 protein, and activity of Caspases-3/8/9, we found that both pathways are involved. Considering that ROS-induced oxidative stress may be the main reason of apoptosis, we investigated whether the addition of an antioxidant, N-Acetyl-L-cysteine (NAC), will ameliorate the apoptosis. Interestingly, we found that both intrinsic and extrinsic apoptosis indicators were markedly recovered by adding NAC in culture media, significant remissions were also observed in the cell viability and apoptosis rate. These results suggested that hypoxia-induced apoptosis of SYCF cells may be mediated by the overproduction of ROS, and the antioxidant may be a useful material to relieve cell damage under hypoxia in fish. In general, this study established a marine fish cell line of SYCF cells, which can be used as an in vitro research model of L. polyactis and for revealing the mechanisms of ROS-induced apoptosis under hypoxic conditions in this species. This provides important insights into the cellular physiology of marine fish under hypoxia.

Anti-Apoptotic c-FLIP Reduces the Anti-Tumour Activity of Chimeric Antigen Receptor T Cells.

Simple SummaryChimeric antigen receptor (CAR) T cell treatment is a promising adoptive cell therapy that utilises CAR-expressing primary T cells to target specific tumour antigens. Since its first approval in 2017, the FDA has approved six CAR T cell therapies for blood cancer treatment. Despite their success in treating B cell malignancies, solid-tumour-directed CAR T cells face multifactorial challenges, often resulting in a lack of anti-tumour response in tumour elimination. The aim of this study was to evaluate the potency of expressing anti-apoptotic gene FLICE-like inhibitory protein p43 (c-FLIPp43) in Her2-CAR T cells to resist activation-induced cell death (AICD). This study confirmed the anti-apoptotic activity of c-FLIPp43. However, the expression of c-FLIPp43 reduced the CAR activity in a Her-2 breast cancer xenograft model relative to the control Her2-CAR T cells. This work provides insight into the role of c-FLIP in T cells and its impact on anti-tumour immunity in CAR T cells.CAR T cell treatment of solid tumours is limited by poor persistence partly due to CD95 ligand (CD95L)-induced apoptosis. Both T cells and cells within the tumour microenvironment (TME) may express CD95L, triggering apoptosis in CD95-receptor-positive CAR T cells. Tonic signalling of CAR T cells may also increase CD95-dependent AICD. Because the intracellular protein c-FLIP protects T cells from AICD, we expressed c-FLIPp43 within a Her-2 targeted CAR cassette and evaluated the potential of c-FLIPp43 through in vitro functional assays and in vivo tumour-bearing xenograft model. cFLIP expression protected against CD95L-induced cell death in the Jurkat T cell lines. However, in primary human CAR T cells containing CAR-CD28 domains, c-FLIPp43 overexpression had minimal additional impact on resistance to CD95L-induded cell death. In vitro cytotoxicity against a breast cancer tumour cell line was not altered by c-FLIPp43 expression, but the expression of c-FLIPp43 in Her2-CAR T cells lowered interferon-γ secretion, without markedly affecting IL-2 levels, and c-FLIPp43-Her2-CAR T cells showed reduced anti-tumour activity in immunodeficient mice with breast cancer. The findings of this study provide a new understanding of the effects of controlling extrinsic apoptosis pathway suppression in CAR T cells, suggesting that c-FLIPp43 expression reduces anti-tumour immunity through the modulation of effector T cell pathways.

Programmed cell death and its role in SARS-CoV-2 infection

Apoptosis is a type of programmed cell death that plays a vital role in removing the redundant cells, damaged cells, or virally infected cells to help maintain homeostasis in tissues. There are three initiating pathways of apoptosis activation including the intrinsic via mitochondrial, extrinsic via death receptor, and perforin/granzyme pathway. Apoptosis is considered as a protective mechanism in the host but when this disordered program causes pathological outcomes. In SARS-CoV-2 virus infection, extrinsic and intrinsic pathways of apoptosis are associated with ORF3a and 7a proteins, respectively. Many studies showed that inappropriate apoptosis is involved in different pathogenicity and severity of COVID-19. Therefore, understanding apoptosis mechanism helps us to have new therapeutic strategies in the treatment of SARSCoV- 2 virus infection and be able to decrease the damage caused by excessive immune responses Key words: programmed cell death, apoptosis, caspase, SARS-CoV-2, COVID-19.

FAIM Enhances the Efficacy of Mesenchymal Stem Cell Transplantation by Inhibiting JNK-Induced c-FLIP Ubiquitination and Degradation.

Background The poor survival rates of transplanted mesenchymal stem cells (MSCs) in harsh microenvironments impair the efficacy of MSCs transplantation in myocardial infarction (MI). Extrinsic apoptosis pathways play an important role in the apoptosis of transplanted MSCs, and Fas apoptosis inhibitory molecule (FAIM) is involved in regulation of the extrinsic apoptosis pathway. Thus, we aimed to explore whether FAIM augmentation protects MSCs against stress-induced apoptosis and thereby improves the therapeutic efficacy of MSCs. Methods We ligated the left anterior descending coronary artery (LAD) in the mouse heart to generate an MI model and then injected FAIM-overexpressing MSCs (MSCsFAIM) into the peri-infarction area in vivo. Moreover, FAIM-overexpressing MSCs were challenged with oxygen, serum, and glucose deprivation (OGD) in vitro, which mimicked the harsh microenvironment that occurs in cardiac infarction. Results FAIM was markedly downregulated under OGD conditions, and FAIM overexpression protected MSCs against OGD-induced apoptosis. MSCsFAIM transplantation improved cell retention, strengthened angiogenesis, and ameliorated heart function. The antiapoptotic effect of FAIM was mediated by cellular-FLICE inhibitory protein (c-FLIP), and FAIM augmentation improved the protein expression of c-FLIP by reducing ubiquitin–proteasome-dependent c-FLIP degradation. Furthermore, FAIM inhibited the activation of JNK, and treatment with the JNK inhibitor SP600125 abrogated the reduction in c-FLIP protein expression caused by FAIM silencing. Conclusions Overall, these results indicated that FAIM curbed the JNK-mediated, ubiquitination–proteasome-dependent degradation of c-FLIP, thereby improving the survival of transplanted MSCs and enhancing their efficacy in MI. This study may provide a novel approach to strengthen the therapeutic effect of MSC-based therapy.

In Vitro and In Vivo Relevant Antineoplastic Activity of Platinum(II) Complexes toward Triple-Negative MDA-MB-231 Breast Cancer Cell Line.

Two platinum complexes [Pt(HL3)Cl]·H2O (3) and [Pt(HL4)Cl]·H2O (4) containing α- and β-naphthyl groups, respectively, were investigated in more detail in vitro and in vivo for antineoplastic activity. The cytotoxicity activity induced by these platinum(II) compounds against breast cancer (MDA-MB-231 and MCF-7), lung (A549), prostate (PC3), pancreas (BXPC-3), and normal peripheral blood mononuclear (PBMC) cells were evaluated by MTT assay. The cell viability MTT assay showed that complex (4) was more cytotoxic to all cancer cell lines tested and less cytotoxic against human PBMC. Therefore, complex (4) was selected to further investigate the mechanism of cytotoxic effects involved against MDA-MB-231 cell line (human triple-negative breast cancer). Sub-G1 analysis of the cell cycle showed that this complex induces cell death by apoptosis due to the cell loss of DNA content detected in flow cytometry. The cytotoxic effect induced by complex (4) was associated with the capability of the complex to induce mitochondrial membrane depolarization, as well as increase ROS levels and caspase activation, as a result of the activation of both extrinsic and intrinsic apoptosis pathways. Ultrastructural alterations were observed using scanning and transmission electron microscopy (SEM and TEM), such as membrane blebbing, filopodia reduction, empty mitochondrial matrix, and DNA fragmentation. Furthermore, complex (4) was tested in an MDA-MB-231 tumor nodule xenograft murine model and demonstrated a remarkable reduction in tumor size in BALB/c nude mice, when compared to the control animals.

Exploring the recent trends in perturbing the cellular signaling pathways in cancer by natural products.

Cancer is commonly thought to be the product of irregular cell division. According to the World Health Organization (WHO), cancer is the major cause of death globally. Nature offers an abundant supply of bioactive compounds with high therapeutic efficacy. Anticancer effects have been studied in a variety of phytochemicals found in nature. When Food and Drug Administration (FDA)-approved anticancer drugs are combined with natural compounds, the effectiveness improves. Several agents have already progressed to clinical trials based on these promising results of natural compounds against various cancer forms. Natural compounds prevent cancer cell proliferation, development, and metastasis by inducing cell cycle arrest, activating intrinsic and extrinsic apoptosis pathways, generating reactive oxygen species (ROS), and down-regulating activated signaling pathways. These natural chemicals are known to affect numerous important cellular signaling pathways, such as NF-B, MAPK, Wnt, Notch, Akt, p53, AR, ER, and many others, to cause cell death signals and induce apoptosis in pre-cancerous or cancer cells without harming normal cells. As a result, non-toxic “natural drugs” taken from nature’s bounty could be effective for the prevention of tumor progression and/or therapy of human malignancies, either alone or in combination with conventional treatments. Natural compounds have also been shown in preclinical studies to improve the sensitivity of resistant cancers to currently available chemotherapy agents. To summarize, preclinical and clinical findings against cancer indicate that natural-sourced compounds have promising anticancer efficacy. The vital purpose of these studies is to target cellular signaling pathways in cancer by natural compounds.