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Background: During adolescence, individuals experience rapid changes in physical, cognitive, emotional, behavioral, and social aspects of their life. Due to certain risks which arise during the developmental period of adolescence, substance use and addiction are encountered as a public health problem. Studies on biochemical parameters and puberty are limited. In this study, adolescents with substance use disorder were compared to healthy individuals in terms of sociodemographic characteristics and serum irisin and caspase-9 levels. Methods: The study group consisted of 32 adolescents with substance use disorder and the control group was comprised of 29 healthy adolescents. The sociodemographic characteristics and serum irisin and caspase-9 levels of the groups were compared. Results: The study group performed lower than the control group in terms of duration of education and continuing education. Serum caspase-9 levels were found to be significantly higher in the study group compared to the control group. There was no difference in irisin levels of the groups. Conclusions: It was determined that substance use negatively affects educational aspects and may also lead to certain destructive biological effects in adolescents. In order to reveal the effect of substance use on adolescents in more detail, studies with larger samples are needed. [ABSTRACT FROM AUTHOR]

Recent advances in whole genome sequencing have revealed an immense microbial potential for the production of therapeutic small molecules, even from well‐known producers. To access this potential, we subjected prominent antimicrobial producers to alternative antiproliferative assays using persistent cancer cell lines. Described herein is our discovery of hirocidins, novel secondary metabolites from Streptomyces hiroshimensis with antiproliferative activities against colon and persistent breast cancer cells. Hirocidin A is an unusual nine‐membered carbocyclic maleimide and hirocidins B and C are relatives with an unprecedented, bridged azamacrocyclic backbone. Mode of action studies show that hirocidins trigger mitochondrion‐dependent apoptosis by inducing expression of the key apoptotic effector caspase‐9. The discovery of new cytotoxins contributes to scaffold diversification in anticancer drug discovery and the reported modes of action and concise total synthetic route for variant A set the stage for unraveling specific targets and biochemical interactions of the hirocidins. [ABSTRACT FROM AUTHOR]

Medicinal plants produce various bioactive molecules with potential anti-cancer properties with favorable safety profiles. We aimed to investigate the comprehensive composition of Vernonia amygdalina leaf extract and its cytotoxic effects via apoptosis in HeLa cells. The metabolomics approach using LC-MS/MS was conducted to gather the metabolite profile of the extract. Proteomics was performed to understand the comprehensive mechanistic pathways of action. The apoptosis was visualized by cellular staining and the apoptotic proteins were evaluated. V. amygdalina leaf extract exhibited dose-dependent cytotoxic effects on both HeLa and Vero cells after 24 h of exposure in the MTT assay with the IC50 values of 0.767 ± 0.0334 and 4.043 ± 0.469 µg mL−1, respectively, which demonstrated a higher concentration required for Vero cell cytotoxicity. The metabolomic profile of 112 known metabolites specified that the majority of them were alkaloids, phenolic compounds, and steroids. Among these metabolites, deacetylvindoline and licochalcone B were suggested to implicate cytotoxicity. The cytotoxic pathways involved the response to stress and cell death which was similar to doxorubicin. The upstream regulatory proteins, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and X-box binding protein 1 (XBP1), were significantly altered, supporting the regulation of apoptosis and cell death. The levels of apoptotic proteins, c-Jun N-terminal kinases (JNK), p53, and caspase-9 were significantly increased. The novel insights gained from the metabolomic profiling and proteomic pathway analysis of V. amygdalina leaf extract have identified crucial components related to apoptosis induction, highlighting its potential to develop future chemotherapy. [ABSTRACT FROM AUTHOR]

Abstract Background Hepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-β signaling pathway may be associated with tumor progression. However, the relationship between TGF-β signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-β signaling pathway on HCC progression as well as the potential regulatory mechanism involved. Methods We conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni–NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo. Results We found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is −1032 to −1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis. Conclusion This study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-β signaling pathway and HCC, which contributes to discover the mechanism of HCC development.

Background: Hepatocellular carcinoma (HCC) represents one of the most significant causes of mortality due to cancer-related deaths. It has been previously reported that the TGF-β signaling pathway may be associated with tumor progression. However, the relationship between TGF-β signaling pathway and HCC remains to be further elucidated. The objective of our research was to investigate the impact of TGF-β signaling pathway on HCC progression as well as the potential regulatory mechanism involved. Methods: We conducted a series of bioinformatics analyses to screen and filter the most relevant hub genes associated with HCC. E. coli was utilized to express recombinant protein, and the Ni–NTA column was employed for purification of the target protein. Liquid liquid phase separation (LLPS) of protein in vitro, and fluorescent recovery after photobleaching (FRAP) were utilized to verify whether the target proteins had the ability to drive force LLPS. Western blot and quantitative real-time polymerase chain reaction (qPCR) were utilized to assess gene expression levels. Transcription factor binding sites of DNA were identified by chromatin immunoprecipitation (CHIP) qPCR. Flow cytometry was employed to examine cell apoptosis. Knockdown of target genes was achieved through shRNA. Cell Counting Kit-8 (CCK-8), colony formation assays, and nude mice tumor transplantation were utilized to test cell proliferation ability in vitro and in vivo. Results: We found that Smad2/3/4 complex could regulate tyrosine aminotransferase (TAT) expression, and this regulation could relate to LLPS. CHIP qPCR results showed that the key targeted DNA binding site of Smad2/3/4 complex in TAT promoter region is −1032 to −1182. In addition. CCK-8, colony formation, and nude mice tumor transplantation assays showed that Smad2/3/4 complex could repress cell proliferation through TAT. Flow cytometry assay results showed that Smad2/3/4 complex could increase the apoptosis of hepatoma cells. Western blot results showed that Smad2/3/4 complex would active caspase-9 through TAT, which uncovered the mechanism of Smad2/3/4 complex inducing hepatoma cell apoptosis. Conclusion: This study proved that Smad2/3/4 complex could undergo LLPS to active TAT transcription, then active caspase-9 to induce hepatoma cell apoptosis in inhibiting HCC progress. The research further elucidate the relationship between TGF-β signaling pathway and HCC, which contributes to discover the mechanism of HCC development. [ABSTRACT FROM AUTHOR]

• Five bivalent Smac mimetics are designed and synthesised. • Mimetics are evaluated in silico with the TwistDock workflow. • Compound 3 among the five displays the highest binding potency to BIR2 and BIR3. • Experiments with SK-OV-3 and MDA-MB-231 cancer cell lines confirm the prediction. Bivalent Smac mimetics have been shown to possess binding affinity and pro-apoptotic activity similar to or more potent than that of native Smac, a protein dimer able to neutralize the anti-apoptotic activity of an inhibitor of caspase enzymes, XIAP, which endows cancer cells with resistance to anticancer drugs. We design five new bivalent Smac mimetics, which are formed by various linkers tethering two diazabicyclic cores being the IAP binding motifs. We built in silico models of the five mimetics by the TwistDock workflow and evaluated their conformational tendency, which suggests that compound 3 , whose linker is n -hexylene, possess the highest binding potency among the five. After synthesis of these compounds, their ability in tumour cell growth inhibition and apoptosis induction displayed in experiments with SK-OV-3 and MDA-MB-231 cancer cell lines confirms our prediction. Among the five mimetics, compound 3 displays promising pro-apoptotic activity and deserves further optimization. [ABSTRACT FROM AUTHOR]

Breast cancer is the second leading contributor to the age-standardized mortality rate, for both sexes and all ages worldwide. In Europe and the United States, it is the second leading cause of mortality, with an incidence rate of about 2.6 million cases per year. Noscapine, a well-known alkaloid used as a cough suppressant, demonstrated anti-tumor effects by triggering apoptosis in various cancer cell lines and has the potential to become another ally against breast, ovarian, colon, and gastric cancer, among other types of malignancy. Apoptosis plays a crucial role in the treatment of cancer. Noscapine affected BAX, CASP8, CASP9, NFKBIA, and RELA gene and protein expression in the MCF-7 and MDA-MB-231 cell lines. Gene expression was higher in tumor than in normal tissue, including the BAX expression levels in lung, ovary, endometrium, colon, stomach, and glioblastoma patients; BCL2L1 expression in endometrium, colon, and stomach patients; CASP8 gene expression levels in lung, endometrium, colon, stomach, and glioblastoma patients; RELA in colon, stomach, and glioblastoma patients; and NFKBIA in glioblastoma patients. It can be concluded that noscapine affected genes and proteins related to apoptosis in cancer cell lines and several types of cancer patients. [ABSTRACT FROM AUTHOR]

Mitochondrial apoptotic signaling cascades lead to the formation of the apoptosome, a 1.1-MDa heptameric protein scaffold that recruits and activates the caspase-9 protease. Once activated, caspase-9 cleaves and activates downstream effector caspases, triggering the onset of cell death through caspase-mediated proteolysis of cellular proteins. Failure to activate caspase-9 enables the evasion of programmed cell death, which occurs in various forms of cancer. Despite the critical apoptotic function of caspase-9, the structural mechanism by which it is activated on the apoptosome has remained elusive. Here, we used a combination of methyl-transverse relaxation-optimized NMR spectroscopy, protein engineering, and biochemical assays to study the activation of caspase-9 bound to the apoptosome. In the absence of peptide substrate, we observed that both caspase-9 and its isolated protease domain (PD) only very weakly dimerize with dissociation constants in the millimolar range. Methyl-NMR spectra of isotope-labeled caspase-9, within the 1.3-MDa native apoptosome complex or an engineered 480-kDa apoptosome mimic, reveal that the caspase-9 PD remains monomeric after recruitment to the scaffold. Binding to the apoptosome, therefore, organizes caspase-9 PDs so that they can rapidly and extensively dimerize only when substrate is present, providing an important layer in the regulation of caspase-9 activation. Our work highlights the unique role of NMR spectroscopy to structurally characterize protein domains that are flexibly tethered to large scaffolds, even in cases where the molecular targets are in excess of 1 MDa, as in the present example. [ABSTRACT FROM AUTHOR]

Apoptosis plays a pivotal role in the immune response to combat pathogen infections. In mammals, caspase-9, abbreviated as Casp9, plays an irreplaceable role in the initiation phase of the apoptotic cascade. To investigate the role of Casp9 in teleosts, we conducted a functional characterization of Casp9 in goldfish (Carassius auratus L.). The open reading frame of GfCasp9 spans 1296 base pairs (bp), encoding a protein composed of 431 amino acids. GfCasp9 was ubiquitously expressed in various tissues, with the spleen and brain showing the highest levels of expression. Subcellular localization analysis revealed that GfCasp9 is distributed in both the cytoplasm and nucleus. Overexpressing of GfCasp9 in HEK293 cells elicits a robust apoptotic response. Additionally, infection with Aeromonas hydrophila significantly increases the mRNA and protein expression of GfCasp9. These findings underscore the critical importance of GfCasp9 in immune responses and apoptosis against bacterial infections. • GfCasp9 mRNA expression was highly expressed in the spleen and brain. • Subcellular localization analysis revealed that GfCasp9 is distributed in both the cytoplasm and nucleus. • Infection with Aeromonas hydrophila significantly increases the mRNA and protein expression of GfCasp9. • Overexpressing of GfCasp9 in HEK293 cells elicits a robust apoptotic response. [ABSTRACT FROM AUTHOR]

Objectives of the Study: The aim of this study is to investigate whether fisetin can effectively reduce the myocardial damage induced by patulin. This study also aims to reveal the mechanism and target of fisetin in inhibiting myocardial damage. Materials and Methods: Network pharmacology was used to screen the targets of fisetin on myocardial damage and the regulatory network of active ingredients-drug targets was constructed. GO and KEGG enrichment analyses were performed to screen out the key pathways and targets of fisetin on myocardial damage. Patulin induced apoptosis in H9c2 cardiomyocytes to verify the key targets. The mechanism of fisetin in inhibiting myocardial damage was determined. Results: FIS can reduce the apoptosis of cardiomyocytes by protecting cardiomyocytes from PAT injury. According to the results of network pharmacology analysis, combined with enzyme activity detection and WB experiment, it was found that the mechanism of FIS to reduce myocardial damage may be related to the P53 signaling pathway, Caspase3/8/9 and Bax/Bcl-2. Conclusion: FIS plays a protective role in PAT-induced myocardial damage. On the one hand, FIS inhibits the protein overexpression of P53, Caspase-9 and Bax. On the other hand, FIS enhances the protein expression of Bcl-2. [ABSTRACT FROM AUTHOR]

Background and Objectives: Natural products have proven to be a valuable source for the discovery of new candidate drugs for cancer treatment. This study aims to investigate the potential therapeutic effects of "Kerra™", a natural extract derived from a mixture of nine medicinal plants mentioned in the ancient Thai scripture named the Takxila Scripture, on HCT116 cells. Materials and Methods: In this study, the effect of the Kerra™ extract on cancer cells was assessed through cell viability assays. Apoptotic activity was evaluated by examining the apoptosis characteristic features. A proteomics analysis was conducted to identify proteins and pathways associated with the extract's mechanism of action. The expression levels of apoptotic protein markers were measured to validate the extract's efficacy. Results: The Kerra™ extract demonstrated a dose-dependent inhibitory effect on the cells, with higher concentrations leading to decreased cell viability. Treatment with the extract for 72 h induced characteristic features of early and late apoptosis, as well as cell death. An LC-MS/MS analysis identified a total of 3406 proteins. The pathway analysis revealed that the Kerra™ extract stimulated apoptosis and cell death in colorectal cancer cell lines and suppressed cell proliferation in adenocarcinoma cell lines through the EIF2 signaling pathway. Upstream regulatory proteins, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and MYC proto-oncogene, bHLH transcription factor (MYC), were identified. The expressions of caspase-8 and caspase-9 were significantly elevated by the Kerra™ extract compared to the chemotherapy drug Doxorubicin (Dox). Conclusions: These findings provide strong evidence for the ability of the Kerra™ extract to induce apoptosis in HCT116 colon cancer cells. The extract's efficacy was demonstrated by its dose-dependent inhibitory effect, induction of apoptotic activity, and modulation of key proteins involved in cell death and proliferation pathways. This study highlights the potential of Kerra™ as a promising therapeutic agent in cancer treatment. [ABSTRACT FROM AUTHOR]

Purpose: Retinal vein occlusion (RVO) is a sight-threatening condition typically treated with intravitreal injection of vascular endothelial growth factor (VEGF) antagonists. Treatment response to anti-VEGF therapies is highly variable, with poor visual outcomes and treatment response in patients with significant retinal nonperfusion following RVO. Recently, caspase-9 has been identified as a potent regulator of edema, gliosis, and neuronal dysfunction during acute retinal hypoxia. The purpose of this study was to compare the therapeutic effect of caspase-9 inhibition against VEGF-neutralization in an established mouse model of RVO. Methods: Adult male C57Bl/6 J mice were randomized to induction of RVO and treatment with either vehicle, intravitreal injection of anti-VEGF antibody, topical administration of a selective caspase-9 inhibitor (Pen1-XBir3), or a combination therapy. Animals were followed on days 1, 2, and 8 after RVO with fundus retinal imaging, and with optical coherence tomography (OCT) to capture retinal swelling, capillary nonperfusion (measured by disorganization of retinal inner layers, DRIL), hyperreflective foci (HRF), and retinal atrophy. Focal electroretinography (ERG) measurements were performed on day 7. Histology was performed on retinal sections from day 8. Results: Both VEGF neutralization and caspase-9 inhibition showed significant retinal protection from RVO compared to vehicle treatment arm. Retinal reperfusion of occluded veins was accelerated in eyes receiving caspase-9 inhibitor, but not significantly different from vehicle in the anti-VEGF group. Retinal edema was suppressed in all treatment groups, with approximately 2-fold greater edema reduction with caspase-9 inhibition compared to VEGF neutralization. HRF were reduced similarly across all treatment groups compared to vehicle. Retinal detachment was reduced only in eyes treated with caspase-9 inhibitor monotherapy. Caspase-9 inhibition reduced retinal atrophy and preserved ERG response; VEGF neutralization did not prevent neurodegeneration following RVO. Conclusion: Caspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in the mouse laser-induced model of RVO. [ABSTRACT FROM AUTHOR]

Thymoquinone (TQ), a polyphenol obtained from Nigella sativa plays a fundamental role in controlling homeostasis. It possesses anti-cancer, anti-oxidant, anti-inflammatory, and analgesic properties. Its anti-cancer properties extend to lymphoma, leukaemia, medulloblastoma, prostate, pancreatic, breast, and ovarian cancer (OvCa). OvCa is one of the most lethal malignancies worldwide having distinct symptoms and a poor prognosis. Resistance to Cisplatin (CDDP) and subsequent reoccurrence of tumors lead to treatment failure. To tackle this, combination therapies along with polyphenols are being studied and have been proven to enhance CDDP response. Certain proteins such as Bcl-2, Bax, STAT3, p53, and caspase-3/9 are involved in cancer progression and signaling of chemoresistance. In this study, Thymoquinone (TQ) interactions with these proteins have been observed through molecular docking. The 3D structure prediction was carried out using Phyre2 and SWISS-MODEL. The molecular docking of protein targets and TQ was done using the PatchDock server. The docked results were then visualized using Discovery Studio. This study helps us to understand the interactions between TQ and apoptotic proteins that help in the augmentation of conventional therapy. This knowledge will also help in developing a new approach and alternate therapeutic strategies using polyphenols like TQ, even in resistant cells. [ABSTRACT FROM AUTHOR]

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, their role in insects is not fully understood; thus, more indepth studies of insect cell apoptosis are necessary. The present study investigates mitochondrial involvement during Conidiobolus coronatus-induced apoptosis in Galleria mellonella hemocytes. Previous research has shown that fungal infection could induce apoptosis in insect hemocytes. Our findings indicate that mitochondria undergo several morphological and physiological changes during fungal infection, e.g., loss of mitochondrial membrane potential, megachannel formation, disturbances in intracellular respiration, increased nonrespiratory oxygen consumption in mitochondria, decreased ATP-coupled oxygen consumption and increased non-ATP–coupled oxygen consumption, decreased extracellular and intracellular oxygen consumption, and increased extracellular pH. Our findings confirm that G. mellonella immunocompetent cells demonstrate Ca2+ overload in mitochondria, translocation of cytochrome c-like protein from mitochondrial to cytosol fraction, and higher activation of caspase-9-like protein after C. coronatus infection. Most importantly, several of the changes observed in insect mitochondria are similar to those accompanying apoptosis in mammalian cells, suggesting that the process is evolutionarily conserved. [ABSTRACT FROM AUTHOR]

PurposeRetinal vein occlusion (RVO) is a sight-threatening condition typically treated with intravitreal injection of vascular endothelial growth factor (VEGF) antagonists. Treatment response to anti-VEGF therapies is highly variable, with poor visual outcomes and treatment response in patients with significant retinal nonperfusion following RVO. Recently, caspase-9 has been identified as a potent regulator of edema, gliosis, and neuronal dysfunction during acute retinal hypoxia. The purpose of this study was to compare the therapeutic effect of caspase-9 inhibition against VEGF-neutralization in an established mouse model of RVO.MethodsAdult male C57Bl/6 J mice were randomized to induction of RVO and treatment with either vehicle, intravitreal injection of anti-VEGF antibody, topical administration of a selective caspase-9 inhibitor (Pen1-XBir3), or a combination therapy. Animals were followed on days 1, 2, and 8 after RVO with fundus retinal imaging, and with optical coherence tomography (OCT) to capture retinal swelling, capillary nonperfusion (measured by disorganization of retinal inner layers, DRIL), hyperreflective foci (HRF), and retinal atrophy. Focal electroretinography (ERG) measurements were performed on day 7. Histology was performed on retinal sections from day 8.ResultsBoth VEGF neutralization and caspase-9 inhibition showed significant retinal protection from RVO compared to vehicle treatment arm. Retinal reperfusion of occluded veins was accelerated in eyes receiving caspase-9 inhibitor, but not significantly different from vehicle in the anti-VEGF group. Retinal edema was suppressed in all treatment groups, with approximately 2-fold greater edema reduction with caspase-9 inhibition compared to VEGF neutralization. HRF were reduced similarly across all treatment groups compared to vehicle. Retinal detachment was reduced only in eyes treated with caspase-9 inhibitor monotherapy. Caspase-9 inhibition reduced retinal atrophy and preserved ERG response; VEGF neutralization did not prevent neurodegeneration following RVO.ConclusionCaspase-9 inhibition confers stronger neuronal and vascular protection compared to VEGF neutralization in the mouse laser-induced model of RVO.

Background: One of the most noteworthy concerns in cancer theranostics in recent decades is the outcome of unique nanoparticles for diagnosing and treating purposes. In the current study, we used unripe Garcinia cambogia fruits to create silver nanoparticles utilizing a straightforward and environmentally friendly process. Materials and Methods: When exposed to fruit extract, the hydroalcoholic silver ions were decreased and stabilized over an extended period, leading to the biogenesis of silver nanoparticles with functionalized surfaces. These organically produced silver nanoparticles' properties were examined and characterized. Results: By measuring mitochondrial membrane potential, formation of the reactive oxygen species, the viability of the cell, and activities of caspase 3 and 9 against the MDA-MB-231 (Breast Cancer Cells), we were able to demonstrate the anti-cancer effects of these nanoparticles in vitro. The findings imply that the AgNPs have cytotoxic and apoptotic characteristics. Conclusion: According to the current research, AgNPs may aid in the creation of an effective anti-cancer drug, which could result in the creation of remarkable nanomedicine for treating different types of cancer. [ABSTRACT FROM AUTHOR]

Objective To investigate the effect of microRNA-15b (miR-15b) gene interference on cerebral ischemiareperfusion injury and its mechanism. Methods Astrocytes from cerebral cortex of newborn Wistar rats within 24 hours of birth were cultured and identified. Oxygen glucose deprivation/recovery method was used to treat the simulated cerebral ischemia-reperfusion injury in vivo, which was recorded as the model group. miR-15b interfering adenovirus vector and negative control vector were constructed and transfected into cells of the above model group respectively, which were recorded as the overexpression group, the silence group, the overexpression control group and the silence control group. Astrocytes from the cerebral cortex without special treatment were used as the blank control group. After 24 hours, the morphological changes of cells were observed by inverted phase contrast microscope in each group. The cell viability was detected by MTT assay, and the cell viability was detected by lactate dehydrogenase (LDH) leakage rate. The apoptosis rate was detected by flow cytometry. Real-time quantitative polymerase chain reaction was used to detect the expression levels of miR-15b, blymphoma-2 (Bcl-2), cysteine protease-3 (Caspase-3) and cysteine protease-9 (Caspase-9) messenger RNA (mRNA). The expressions of Bcl-2, Caspase-3 and Caspase-9 protein levels were detected by Western blot assay. Luciferase reporter gene experiment verified that miR-15b could target and regulate Bcl-2. Results Compared with the control group, the adherent cells decreased in the blank group, the overexpression control group and the silence control group, cells shrunk and became round, and the distribution was loose, cell viability and Bcl-2 mRNA and protein expression decreased, cell viability and apoptosis rate, and the expression levels of miR-15b, Caspase-3, Caspase-9 mRNA and protein increased (P＜0.05). Compared with the blank group and the silencing control group, the number of adherent cells in the overexpression group decreased and was more sparsely distributed. It could be seen that cells floated in the culture medium, the cell survival rate and the expression of Bcl-2 mRNA and protein in the overexpression group decreased, the cell viability and apoptosis rate, and the expression of miR-15b, the expressions of Caspase-3, Caspase-9 mRNA and protein increased (P＜0.05). Compared with the blank group and the silenced control group, the adherent cells in the silenced group increased, but still lower than the control group. The cells shrank slightly, the cell survival rate and the expression of Bcl-2 mRNA and protein increased in the silencing group, the cell viability and apoptosis rate, and the expression of miR-15b, the expressions of Caspase-3, Caspase-9 mRNA and protein decreased (P＜0.05). Results of luciferase reporter gene experiments confirmed that miR-15b can target and regulate Bcl-2. Conclusion Overexpression of miR-15b can aggravate cerebral ischemiareperfusion injury, presumably through the mitochondrial pathway to induce the deformation and apoptosis of damaged nerve cells. [ABSTRACT FROM AUTHOR]

Purpose: Spinal cord injury (SCI) is a condition that causes disturbances in normal sensory, motor, and autonomic functions. During SCI, damages occur such as, contusion, compression, distraction. The aim of this study was to investigate effects of the antioxidative thymoquinone on neuron and glia cells in SCI biochemically, immunohistochemically and ultrastructurally. Methods: Male Sprague-Dawley rats were divided into Control, SCI and SCI + Thymoquinone groups. After T10-T11 laminectomy was performed, a metal weight of 15 grams was left down the spinal tube for spinal damage. Immediately after the trauma, the muscles and skin incision were sutured. Thymoquinone was given to the rats by gavage as 30mg/kg/21days. Tissues fixed in 10% formaldehyde, embedded in paraffin wax and immunstained with Caspase-9 and phosphorylated signal transducer and activator of transcription 3 (pSTAT-3) antibodies. Remaining were stored at -80oC for biochemistry. Frozen spinal cord tissues were placed in a phosphate buffer solution and homogenized, centrifuged then used to measure malondialdehyde (MDA) levels, glutathione peroxidase (GSH) and Myeloperoxidase (MPO). Results: In the SCI group, MDA, MPO, neuronal degeneration, vascular dilatation, inflammation, apoptotic appearance in the nucleus, loss of membrane and cristae in mitochondria, and dilatation in the endoplasmic reticulum were observed due to degeneration in the neuron structure. In the electron microscopic examination of the trauma + thymoquinone group, the membranes of the nuclei of the glial cells were thick and euchromatin, and mitochondria were shortened in length. In the SCI group, pyknosis and apoptotic changes were observed in neuronal structures and nuclei of glia cells in the substantia grisea and substantia alba region, along with positive Caspase-9 activity. An increase in Caspase-9 activity was observed in endothelial cells in blood vessels. In the SCI + thymoquinone group, Caspase-9 expression was positive in some of the cells in the ependymal canal while the cuboidal cells showed a negative Caspase-9 reaction in the majority. A few degenerated neurons in the substantia grisea region showed a positive reaction with Caspase-9. In SCI group, pSTAT-3 expression was positive in degenerated ependymal cells, neuronal structures, and glia cells. pSTAT-3 expression was positive in the endothelium and surrounding aggregated cells of the enlarged blood vessels. In the SCI+ thymoquinone group, pSTAT-3 expression was negative in most of the bipolar and multipolar neuron structures and glial cells in ependymal cells, enlarged blood vessel endothelial cells. Conclusions: It has been thought that thymoquinone application in spinal cord injuries may be an antioxidant that can be recommended as an alternative treatment in suppressing the apoptosis of neural cells by significantly reducing the inflammation process. [ABSTRACT FROM AUTHOR]

Objective: As an aggressive type of brain cancer, glioblastoma remains obscure with its short survival time and unclear molecular architecture. Single nucleotide polymorphisms (SNPs), such as those located in the regions of the caspase-9 gene (CASP9), have been reported to be associated with genetic susceptibility to glioblastoma. There is no exact result on the effect of CASP9 SNP rs1052576 on glioblastoma and its biomarker candidacy for the Turkish population. Investigating the polymorphism of the exon 5 (+32 G/A) region of the CASP9 in glioblastoma patients in the Turkish population was the aim of this study. Materials and Methods: Real-time polymerase chain reaction (RT-PCR) method on blood samples of glioblastoma patients (n=33) and healthy controls (n=35) were used to analyze CASP9 SNP rs1052576. Statistical data were obtained using SPSS v.23 software. Results: For CASP9 rs1052576, no evidence was found of its role in glioblastoma (p=0.594). Conclusion: This study was designed to determine the association between glioblastoma and CASP9 SNP rs1052576 within the Turkish population. Our results indicated that CASP9 SNP rs1052576 is not related to glioblastoma in Turkish patients. To clarify these results, further studies with a larger sample size are needed. [ABSTRACT FROM AUTHOR]

Objective: To investigate the effects of Niuhuang (Bovis Calculus, BC) and Shexiang (Moschus) (BC-Moschus) on human hepatocellular carcinoma (HCC) cells SMMC-7721 and a nude mouse model of subcutaneous xenografts, and to explore its anti-HCC mechanism. Methods: The BC-Moschus combination was applied to two liver cancer models in vivo and in vitro. SMMC-7721 was divided into the BC-Moschus group and the control group, and different doses (rude drug dosage 0.625, 1.25, 2.5, and 5 mg/mL) of BC-Moschus extract were used for the intervention. The proliferation ability of HCC cells was detected using the Cell Counting Kit-8 (CCK-8) assay, and the migration ability was detected by a wound healing assay. A subcutaneous xenograft model was prepared using nude mice with human HCC. Specific pathogen-free-grade BALB/c nude mice (5-week-old) were randomly divided into the following groups (n = 6 per group): control (0.9% physiological saline 0.2 mL/d), BC-Moschus [BC 45.5 mg/(kg·d)+ Moschus 13 mg/(kg·d)], and cisplatin (DDP, intraperitoneal injection 5 mg/kg per week) groups. All groups were administered for 14 d. The volume and mass of the subcutaneous xenografts in nude mice were observed. The expression levels of phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway, apoptosis-associated factor p70 S6 Kinase (S6K), Bax, Bcl-2, caspase-3, and caspase-9 in nude mice subcutaneous xenografts were measured by real-time quantitative PCR (RT-qPCR) and Western blot. Terminal Deoxynucleotidy Transferase-Mediated dUTP Nick-End Labeling (TUNEL) was used for quantitative analysis of apoptotic cells. Results: The CCK-8 assay demonstrated that the BC-Moschus combination inhibited HCC cell proliferation in a superior manner to the use of BC and Moschus alone, and the inhibition effect was dose- and time-dependent (P

Hydroquinone (HQ) is one of benzene metabolites that can cause oxidative stress damage and Homologous recombination repair (HR). A good deal of reactive oxygen species (ROS) generated by oxidative stress can trigger apoptotic signaling pathways. The nuclear factor erythroid 2-related factor 2 (Nrf2) can regulate the cell response to oxidative stress damage. The aim of this study was to explore whether Nrf2 participate in HQ-induced apoptosis and its mechanism. The findings displayed that HQ triggered HR, promoted Nrf2 transfer into the cell nucleus and induced cell apoptosis, while Nrf2 deficient elevated cell apoptosis, attenuated the expression of PARP1 and RAD51. We also observed that Nrf2 deficient triggered Caspase-9. Thus, we speculated that Nrf2 might participate in HQ-induced cell apoptosis through Caspase-9 dependent pathways. Meanwhile, Nrf2 participated in HQ-induced DNA damage repair by regulating the level of PARP1 and RAD51. • HQ is a carcinogen, which affects DNA damage and apoptosis in TK6 cells. • Nrf2 is involved in HQ-induced TK6 cells apoptosis through Caspase-9 pathway. • Nrf2 participates in HQ-induced DNA damage repair by regulating PARP1 and RAD51. • Nrf2 may be a potential biomarker of HQ-induced apoptosis and DNA damage repair. [ABSTRACT FROM AUTHOR]

Background & Aims: The 2-pore potassium channel subfamily K member 9 (KCNK9) regulates intracellular calcium concentration and thus modulates cell survival and inflammatory signaling pathways. It also was recognized as a risk allele for inflammatory bowel disease. However, it remains unclear whether KCNK9 modulates inflammatory bowel disease via its impact on immune cell function or whether its influence on calcium homeostasis also is relevant in intestinal epithelial cells. Methods: Kcnk9-/- mice were challenged with 3% dextran sulfate sodium (DSS) to induce experimental acute colitis. Primary cultures of intestinal epithelial cells were generated, and expression of potassium channels as well as cytosolic calcium levels and susceptibility to apoptosis were evaluated. Furthermore, we evaluated whether KCNK9 deficiency was compensated by the closely related 2-pore potassium channel KCNK3 in vivo or in vitro. Results: Compared with controls, KCNK9 deficiency or its pharmacologic blockade were associated with aggravated DSS-induced colitis compared with wild-type animals. In the absence of KCNK9, intestinal epithelial cells showed increased intracellular calcium levels and were more prone to mitochondrial damage and caspase-9–dependent apoptosis. We found that expression of KCNK3 was increased in Kcnk9-/- mice but did not prevent apoptosis after DSS exposure. Conversely, increased levels of KCNK9 in Kcnk3-/- mice were associated with an ameliorated course of DSS-induced colitis. Conclusions: KCNK9 enhances mitochondrial stability, reduces apoptosis, und thus supports epithelial cell survival after DSS exposure in vivo and in vitro. Conversely, its increased expression in Kcnk3-/- resulted in less mitochondrial damage and apoptosis and was associated with beneficial outcomes in DSS-induced colitis.

miR-101a-3p is expressed in a variety of organs and tissues and plays a regulatory role in many diseases, but its role in spinal cord ischemia/reperfusion injury remains unclear. In this study, we established a rat model of spinal cord ischemia/reperfusion injury by clamping the aortic arch for 14 minutes followed by reperfusion for 24 hours. Results showed that miR-101a-3p expression in L4–L6 spinal cord was greatly decreased, whereas MYCN expression was greatly increased. Dual-luciferase reporter assay results showed that miR-101a-3p targeted MYCN. MYCN immunoreactivity, which was primarily colocalized with neurons in L4–L6 spinal tissue, greatly increased after spinal cord ischemia/reperfusion injury. However, intrathecal injection of an miR-101a-3p mimic within 24 hours before injury decreased MYCN, p53, caspase-9 and interleukin-1β expression, reduced p53 immunoreactivity, reduced the number of MYCN/NeuN-positive cells and the number of necrotic cells in L4–L6 spinal tissue, and increased Tarlov scores. These findings suggest that the miR-101a-3p mimic improved spinal ischemia/reperfusion injury-induced nerve cell apoptosis and inflammation by inhibiting MYCN and the p53 signaling pathway. Therefore, miR-101a-3p mimic therapy may be a potential treatment option for spinal ischemia/reperfusion injury.

OBJECTIVE: Ischemia (I) causes lack of oxygen delivery to the tissues, leading to hypoxia and cellular damage. Reperfusion (R) is the re-blooded of the tissue; however, it may cause more tissue damage than ischemia alone in some cases. During IR, number of free radicals and metabolic by-products increases. To prevent this, cellular antioxidant system is activated but it may not be enough to restore the cellular activities. The aim of this study was to investigate the potential protective effect of Ellagic Acid on damage caused by hepatic IR in rats. MATERIALS AND METHODS: Thirty rats were divided into three groups: (1) Sham group – no drug administration but only midline laparotomy was performed in the abdomen; (2) Ischemia-Reperfusion (IR) group – ischemia was applied for 1 hour by sealing the portal vein and hepatic artery, then vessels were reperfused for 6 hours; (3) IR+Elagic Acid (EA) group – after IR, 10 mg/kg of EA was given intraperitonially to the rats once a day for 28 days. Oxidative stress markers were analyzed by blood collection. Hepatic tissues were processed for histological and immunohistochemical analysis. RESULTS: MDA level and MPO activity were increased and GSH content was decreased after IR group was compared to sham group. After EA treatment, these values were improved in IR+EA group. IR caused hepatocyte degeneration, sinusoidal dilatation, leukocyte infiltration and disintegrity of hepatic tissue. EA administration improved histopathology after IR. IR injury increased TNF-α and Caspase-9 expression in hepatocytes and vascular endothelial cells in IR group; however, both decreased in EA-received group. CONCLUSIONS: Ellagic Acid may reduce oxidative stress level and prevent induction of inflammation and cell death against hepatic IR. [ABSTRACT FROM AUTHOR]

Protective effect of royal jelly (RJ) on fluoride-induced nephrotoxicity was investigated in this study. 42 healthy male Wistar rats (n = 42, 8 weeks of age) were divided equally into 6 groups with 7 rats in each; (1) Group-1: Controls fed with standard diet; (2) Group-2: RJ [100 mg/kg] bw (body weight), by oral gavage; (3) Group-3: Fluoride [50 mg/kg] bw, in drinking water; (4) Group-4: Fluoride [100 mg/kg] bw, in drinking water; (5) Group-5: RJ [100 mg/kg] bw, by oral gavage + Fluoride [50 mg/kg] bw, in drinking water; (6) Group-6: RJ [100 mg/kg] bw, by oral gavage + Fluoride [100 mg/kg] bw, in drinking water. After 8 weeks, all rats were decapitated and their kidney tissues were removed for further analysis. The protein expression levels of caspase-3, caspase-6, caspase-9, Bcl-2, Bax, VEGF, GSK-3, BDNF, COX-2 and TNF-α proteins in kidney tissue were analysed by western blotting technique RJ increased Bcl-2, COX-2, GSK-3, TNF-α and VEGF protein levels and a decreased caspase-3, caspase −6, caspase-9, Bax and BDNF protein levels in fluoride-treated rats. RJ application may have a promising therapeutical potential in the treatment of many diseases in the future by reducing kidney damage. [ABSTRACT FROM AUTHOR]

The two new complexes, namely [NiU2(H2O)2]SO4, (NiU) and [CuU2(H2O)2]SO4, (CuU) [U = 5,6-diamino-1,3-dimethyl-2,4(1H,3H)-pyrimidinedione monohydrate] were synthesized and characterized by elemental analysis, physicochemical, spectroscopic, (UV-visible, IR, ESI-MS, and XRD), thermal analysis and cyclic voltametric techniques. As showed in the data obtained, an octahedral geometry has been assigned for the two complexes. The data proved that the new complexes NiU and CuU have anticancer activities against colon cancer (HCT-116), epithelioid carcinoma (HeLa), and prostate adenocarcinoma (PC3) human cancer cell lines. The apoptotic pathway for both NiU and CuU complexes was via p53 elevation, which can arrest the cell cycle and activate the release of cytochrome c. The HCT-116 cells were treated with either NiU or CuU complexes had increased the production of caspase-9, which can activate the production of caspase-3 that degrades all the proteins in the cell, leading to apoptotic cell death. Consequently, the two complexes have promising anticancer activities. [ABSTRACT FROM AUTHOR]

During low tide intertidal organisms face severe temperature fluctuations. This situation may be more pronounced in the context of climate change. To investigate the molecular response mechanism of Onchidium reevesii when stressed by hot and cold temperatures, we cloned its Caspase-3 and Caspase-9 genes and detected their expression characteristics using RT-qPCR. The results showed that the full-length of the cDNA of Caspase-3 consist of 1831 bp, and included an open reading frame (ORF) of 894 bp, a 5′UTR of 155 bp and a 3′UTR of 300 bp, and encoding for 297 amino acids. The full-length of the cDNA of Caspase-9 consisted of 2425 bp, and included an ORF of 1373 bp, a 5′UTR of 290 bp, and a 3′UTR of 759 bp, and encoding for 457 amino acids. During the evolution of species the two genes have been greatly conserved. A homology analysis and phylogenetic tree reconstruction revealed that O. reevesii is most closely related to Aplysia californica. The RT-qPCR showed that both genes were expressed in all of the tissues and that expression was highest in the hepatopancreas. It is speculated that these genes play an essential role in the immunity of O. reevesii to certain diseases. Conditions of temperature stress (40 °C and 10 °C) showed that both heat and cold stresses could induce significant changes in both genes.Compared to the control group, under cold stress conditions the levels of expression of Caspase-3 and Caspase-9 were significantly upregulated (P 0.05) from the control group. When subjected to various stress conditions the two genes displayed different expression patterns, indicating that the species has different strategies for coping with temperature changes. It further indicates that the effect of temperature on the species may play a role through the Caspase-dependent pathway. These may help to better understand the response, at the molecular level, of O. reevesii to conditions of hot and cold stress.

Background and Objectives: Natural products have proven to be a valuable source for the discovery of new candidate drugs for cancer treatment. This study aims to investigate the potential therapeutic effects of “Kerra™”, a natural extract derived from a mixture of nine medicinal plants mentioned in the ancient Thai scripture named the Takxila Scripture, on HCT116 cells. Materials and Methods: In this study, the effect of the Kerra™ extract on cancer cells was assessed through cell viability assays. Apoptotic activity was evaluated by examining the apoptosis characteristic features. A proteomics analysis was conducted to identify proteins and pathways associated with the extract’s mechanism of action. The expression levels of apoptotic protein markers were measured to validate the extract’s efficacy. Results: The Kerra™ extract demonstrated a dose-dependent inhibitory effect on the cells, with higher concentrations leading to decreased cell viability. Treatment with the extract for 72 h induced characteristic features of early and late apoptosis, as well as cell death. An LC-MS/MS analysis identified a total of 3406 proteins. The pathway analysis revealed that the Kerra™ extract stimulated apoptosis and cell death in colorectal cancer cell lines and suppressed cell proliferation in adenocarcinoma cell lines through the EIF2 signaling pathway. Upstream regulatory proteins, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and MYC proto-oncogene, bHLH transcription factor (MYC), were identified. The expressions of caspase-8 and caspase-9 were significantly elevated by the Kerra™ extract compared to the chemotherapy drug Doxorubicin (Dox). Conclusions: These findings provide strong evidence for the ability of the Kerra™ extract to induce apoptosis in HCT116 colon cancer cells. The extract’s efficacy was demonstrated by its dose-dependent inhibitory effect, induction of apoptotic activity, and modulation of key proteins involved in cell death and proliferation pathways. This study highlights the potential of Kerra™ as a promising therapeutic agent in cancer treatment.

Breast cancer is the leading cause of death in women around the world. Medinilla speciosa (Parijoto) contains quercetin, which has been linked to cancer prevention. However, there have been no previous reports on the cytotoxic effect of Medinilla speciosa on resistant breast cancer cells, such as MCF-7/HER-2. The study was therefore conducted to investigate the cytotoxic effects and mechanism of action of Medinilla speciosa methanol fruit extract (MSMFE) on MCF-7/HER-2 cancer cells, both alone and in combination with cisplatin. MCF-7/HER-2 and Vero cell lines were obtained for the in vitro study. Methanol extract was prepared from Medinilla speciosa fruits. Different concentrations of the extract and cisplatin were tested against the MCF-7/HER-2 cells. For the combination treatment, 88 µg/mL of the MSMFE was added to cisplatin in two different concentrations (2.4 and 8 µM). The MTT assay was employed to evaluate the cytotoxic effects of the extract. Cell viability and expression of caspase-9 and VEGF proteins were analyzed. The results showed that the MSMFE had an IC50 value of 175±0.962 µg/mL on MCF-7/HER-2 cells, indicating a cytotoxic effect. After MSMFE administration, there was an increase in caspase-9 protein expression and a decrease in VEGF expression, and the combination of MSMFE and cisplatin lowered cell viability by 53%. The findings of the study revealed that when combined with cisplatin, Medinilla speciosa methanol fruit extract may have synergistic cytotoxic effects on MCF-7/HER-2 cancer cells. It may also induce apoptosis through increased expression of the caspase-9 protein and inhibit angiogenesis by lowering VEGF expression. [ABSTRACT FROM AUTHOR]

Heavy-metal pollution has increasingly jeopardized the habitats of marine organisms including the sea cucumber, a seafloor scavenger vital to seawater bio-decontamination, ocean de-acidification and coral-reef protection. Normal physiology including immune functions of sea cucumbers is toxicologically modulated by marine metal pollutants such as cadmium (Cd). The processes underpinning Cd's toxic effects on immune systems in the sea cucumber, Holothuria leucospilota, are still poorly understood. To this end, we cloned and characterized a full-length caspase-9 (Hl-CASP9) cDNA in the sea cucumber, Holothuria leucospilota. Hl-CASP9 mRNA levels evolved dynamically during embryonic development. Coelomocytes, a type of phagocytic immune effectors central to H. leucospilota immunity, were found to express Hl-CASP9 mRNA most abundantly. Hl-CASP9 protein structurally resembles caspases-2 and -9 in both invertebrate and vertebrate species, comprising a CARD domain and a CASc domain. Remarkably, Hl-CASP9 was transcriptionally sensitive to abiotic oxidative stress inducers including hydrogen peroxide (H2O2), nitric oxide (•NO) and cadmium (Cd), but insensitive to immunostimulants including lipopolysaccharide (LPS), and poly(I:C). Overexpression of Hl-CASP9 augmented mitochondria-dependent apoptosis in HEK293T cells, while knock-down of Hl-CASP9 blunted Cd-induced coelomocyte apoptosis in vivo. Overall, we illustrate that an evolutionarily ancient caspase-9-dependent pathway exists to sensitize coelomocytes to premature cell death precipitated by heavy metal pollutants, with important implications for negative modulation of organismal immune response in marine invertebrates. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder molecularly characterized by the formation of amyloid β (Aβ) plaques and type 2 microtubule-associated protein (Tau) abnormalities. Multiple studies have shown that many of the brain's immunological cells, specifically microglia and astrocytes, are involved in AD pathogenesis. Cells of the innate immune system play an essential role in eliminating pathogens but also regulate brain homeostasis and AD. When activated, innate immune cells can cause programmed cell death through multiple pathways, including pyroptosis, apoptosis, necroptosis, and PANoptosis. The cell death often results in the release of proinflammatory cytokines that propagate the innate immune response and can eliminate Aβ plaques and aggregated Tau proteins. However, chronic neuroinflammation, which can result from cell death, has been linked to neurodegenerative diseases and can worsen AD. Therefore, the innate immune response must be tightly balanced to appropriately clear these AD-related structural abnormalities without inducing chronic neuroinflammation. In this review, we discuss neuroinflammation, innate immune responses, inflammatory cell death pathways, and cytokine secretion as they relate to AD. Therapeutic strategies targeting these innate immune cell death mechanisms will be critical to consider for future preventive or palliative treatments for AD. [ABSTRACT FROM AUTHOR]

Liposarcoma is a rare and heterogeneous soft tissue malignant tumor and has a significant impact on mortality with a poor prognosis. To date, there is no effective treatment for liposarcoma, whereas surgical resection is only the gold treatment with numerous adverse effects. Here we investigated whether tetrandrine inhibits the growth of SW872 human malignant liposarcoma cells. Of note, tetrandrine at 10 μM vastly inhibited growth and induced apoptosis, as evidenced by increased nuclear DNA fragmentation and sub-G1 population of SW872 cells. Mechanistically, treatment with tetrandrine led to activation of caspase-9/3 in SW872 cells, and z-VAD-fmk, a pan-caspase inhibitor, attenuated the tetrandrine-induced apoptosis and growth suppression in SW872 cells. In addition, tetrandrine treatment resulted in down-regulation of XIAP andSTAT-3 in SW872 cells, and importantly knockdown of STAT-3 caused a significant reduction of the cell survival. Tetrandrine also had abilities to up-regulate not only the expression of GRP78 and ATF-4 but also the phosphorylation of eIF-2α in SW872 cells. In summary, these results demonstrated that tetrandrine has strong growth-suppressive and apoptosis-inducing effects on SW872 cells, which are mediated through control of the intrinsic caspase pathway, down-regulation of XIAP and STAT-3, and triggering ER stress. [ABSTRACT FROM AUTHOR]

Abstract Background Glufosfamide (β-d-glucosylisophosphoramide mustard, GLU) is an alkylating cytotoxic agent in which ifosforamide mustard (IPM) is glycosidically linked to the β-d-glucose molecule. GLU exerted its cytotoxic effect as a targeted chemotherapy. Although, its cytotoxic efficacy in a number of cell lines, there were no experimental or clinical data available on the oncolytic effect of oxazaphosphorine drugs in hepatocellular carcinoma. Therefore, the main objective of the current study is to assess the cytotoxic potential of GLU for the first time in the hepatocellular carcinoma HepG2 cell line model. Methods Cytotoxicity was assayed by the MTT method, and half-maximal inhibitory concentration (IC50) was calculated. Flow cytometric analysis of apoptosis frequencies was measured by using Annexin V/PI double stain, an immunocytochemical assay of caspase-9, visualization of caspase-3, and Bcl2 gene expression were undertaken as apoptotic markers. Mitochondrial membrane potential was measured using the potentiometric dye; JC-1, as a clue for early apoptosis as well as ATP production, was measured by the luciferase-chemiluminescence assay. Results Glufosfamide induced cytotoxicity in HepG2 cells in a concentration- and time-dependent manner. The IC50 values for glufosfamide were significantly lower compared to ifosfamide. The frequency of apoptosis was much higher for glufosfamide than that of ifosfamide. The contents of caspase-9 and caspase-3 were elevated following exposure to GLU more than IFO. The anti-apoptotic Bcl2 gene expression, the mitochondrial membrane potential, and the cellular ATP levels were significantly decreased than in case of ifosfamide. Conclusions The current study reported for the first time cytotoxicity activity of glufosfamide in HepG2 cells in vitro. The obtained results confirmed the higher oncolytic activity of glufosfamide than its aglycone ifosfamide. The generated data warrants further elucidations by in vivo study.

Abstract Background Monosodium glutamate (MSG) is frequently consumed as a flavor enhancer or food additive. Possible damages induced by MSG effects on some organs have been stated in experimental animal models. The aim of the present study was to evaluate the protective effects of L-carnitine (L-ca) on the renal tissue in MSG-Induced Rats. Methods In this regard, 60 male rats were randomly divided into six groups (n = 10/each): 1 (Control); 2 (sham); 3 (L-carnitine 200 mg/kg b.w); 4 (MSG 3 g/kg b.w); 5 (MSG + L-carnitine 100 mg/kg); and 6 (MSG + L-carnitine 200 mg/kg). After 6 months, the rats were sacrificed, the blood sample collected and the kidneys harvested for evaluation of biochemical analytes, genes expression, and histopathological changes. Results MSG significantly increased the serum level of MDA, BUN, creatinine, uric acid and renal Caspase-9, NGAL and KIM-1 expression, but it decreased the serum activity also renal expression of SOD, catalase, GPX, and Bcl-2 expression compared to the control group. Treatment with L-ca significantly reduced the serum BUN, creatinine, uric acid and MDA level and increased catalase, GPX and SOD compared to the MSG group. However, only administration of L-ca 200 significantly decreased the caspase-9, NGAL and KIM-1; also, it increased the Bcl-2 expression in the kidney compared to the MSG group. Conclusions Our findings indicated that L-carnitine had a major impact on the cell protection and might be an effective therapy in ameliorating the complications of the kidney induced by MSG via its antioxidant and anti-apoptotic properties.

Besides cell death, caspase-9 participates in non-apoptotic events, including cell differentiation. To evaluate a possible impact on the expression of chondrogenic/osteogenic factors, a caspase-9 inhibitor was tested in vitro. For this purpose, mouse forelimb-derived micromass cultures, the most common chondrogenic in vitro model, were used. The following analyses were performed based on polymerase chain reaction (PCR) arrays and real-time PCR. The expression of several chondrogenesis-related genes was shown to be altered, some of which may impact chondrogenic differentiation (Bmp4, Bmp7, Sp7, Gli1), mineral deposition (Alp, Itgam) or the remodelling of the extracellular matrix (Col1a2, Mmp9) related to endochondral ossification. From the cluster of genes with altered expression, Mmp9 showed the most significant decrease in expression, of more than 50-fold. Additionally, we determined the possible impact of caspase-9 downregulation on the expression of other Mmp genes. A mild increase in Mmp14 was observed, but there was no change in the expression of other studied Mmp genes (-2, -3, -8, -10, -12, -13). Interestingly, inhibition of Mmp9 in micromasses led to decreased expression of some chondrogenic markers related to caspase-9. These samples also showed a decreased expression of caspase-9 itself, suggesting a bidirectional regulation of these two enzymes. These results indicate a specific impact of caspase-9 inhibition on the expression of Mmp9. The localisation of these two enzymes overlaps in resting, proliferative and pre-hypertrophic chondrocytes during in vivo development, which supports their multiple functions, either apoptotic or non-apoptotic. Notably, a coincidental expression pattern was identified in Pik3cg, a possible candidate for Mmp9 regulation. [ABSTRACT FROM AUTHOR]

β-Glucans (βG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of βG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. βG was not cytotoxic. The mRNA levels of CCNA2 of cells exposed to β-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9 suggest that βG could inhibit promotion and progression steps of carcinogenesis, eliminating neoplastic cells. The upregulation of CCNA2 gene in combined treatment could be occurred due to ability of βG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1 suggests that βG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of βG during chemotherapy

The quinazoline nucleus is one of the most privileged scaffolds for the designing of pharmacodynamic compounds.There were several molecules originated from natural as well as synthetic sources.Molecules with quinazolinenucleus have been found not only aspotential anticancer agents butalsofor treating other diseases.In the present study, the designed compounds were subjected toin silico screening followed by molecular docking with caspase-9 protein. Initially, compounds were screened for drug-likeness usingSwissADME, Osiris Property Explorer, ProTox-II, and PASS web servers.It has been found that most of the compounds interactwith caspase-9 through H-bonding. During the preliminary screening, the compounds have shown druglikeness anddevoid of any mutagenicity and hepatotoxicity.Our results indicate that these scaffolds could be further improved for their anticancer efficacy by structural modifications. [ABSTRACT FROM AUTHOR]

ß-Glucans (ßG) are polysaccharides widely distributed in nature with chemopreventive properties. The aim of this study was to investigate the effects of ßG and the combined treatment with doxorubicin (Dox) on cell viability and mRNA levels of genes involved in cell cycle, apoptosis and antioxidant response. ßG was not cytotoxic. The mRNA levels of CCNA2 of cells exposed to ß-glucan was upregulated and the exposure to Dox decreased the expression, while the combination led to an upregulation. Modulation of mRNA levels of CASP9 suggest that ßG could inhibit promotion and progression steps of carcinogenesis, eliminating neoplastic cells. The upregulation of CCNA2 gene in combined treatment could be occurred due to ability of ßG in restoring the cell cycle distribution pattern after treatment with Dox. The upregulation of SOD1 suggests that ßG can enhance the intracellular antioxidant defense, reducing the levels of superoxide dismutase induced by Dox. This response could reduce oxidative damage and attenuate tissue damage during chemotherapeutic treatment. Our data suggest that the drug combination may be less effective in killing tumor cells than the treatment with Dox alone. Thus, future studies should carefully consider this effect on indication of ßG during chemotherapy. [ABSTRACT FROM AUTHOR]

Caspase-9 (CASP9) and caspase-10 (CASP10) polymorphisms were associated with human cancers; however, the results remain controversial. In this meta-analysis, we aimed to estimate the relationship among CASP9 (rs1052576, rs1052571, rs4645978, rs4645981, rs4645982, rs2308950) and CASP10 (rs13006529, rs13010627, rs3900115) polymorphisms and the overall risk of cancers. Relevant studies were obtained from Web of Science, MEDLINE, PubMed, Scopus, and Google scholar databases (updated January 1, 2021). Odds ratio (OR) and 95% confidence intervals (CIs) were measured to estimate the strength of association. Our meta-analysis included 40 studies. The rs4645981 significantly enhanced the risk of cancer under TT vs. CC (OR = 2.42), TC vs. CC (OR = 1.55), TT+ TC vs. CC (OR = 1.66), TT vs. TC + CC (OR = 1.91), and T vs. C (OR = 1.57) inheritance models. As for the rs1052571 variant, increased risk of cancer was observed under TT vs. CC (OR =1.22), TC vs. CC (OR = 1.17), and TT+ TC vs. CC (OR = 1.18) models. The stratified analysis showed a significant correlation between rs4645978 or rs4645981 polymorphisms and cancer risk, while in Asians rs4645978 conferred an increased risk of colorectal, lung, and prostate cancer. Both rs4645981 and rs1052576 polymorphisms were correlated with an enhanced risk of lung cancer. In conclusion, our meta-analysis suggested that CASP9 rs4645981 and rs1052571 polymorphisms are associated with overall cancer risk. More studies on larger populations are warranted to validate these associations. [ABSTRACT FROM AUTHOR]

Background/aim: Hepcidin is the main hormone in the regulation of iron metabolism which is also released from the heart. The aim of our study was to investigate the effects of hepcidin on the cardiac ischemia-reperfusion injury. Materials and methods: In this study, 12 Wistar albino rats were divided into two groups (n = 6 each): 1) The ischemia-reperfusion group (Group 1); 2) Hepcidin-treated group (Group 2). Rat hearts were perfused on Langendorff system with KH (Krebs-Henseleit) and subjected to 30 min stabilization, 30 min global ischemia, and 30 min reperfusion. Hepcidin (- M) was applied to group 2 at the onset of ischemia. Malondialdehyde (MDA), glutathione (GSH), and nitric oxide (NOx) levels were measured in heart tissue for NOx levels, viscosity, and ion content of perfusate were collected before ischemia and the 1st, 5th, 10th, 20th, and 30th minutes of reperfusion were determined. Apoptosis in heart was evaluated. Results: NOx and MDA levels significantly decreased in heart tissue in Hepcidin-treated group. NOx and viscosity of perfusate were not significantly different between the groups. Perfusate iron, calcium, magnesium, potassium, and sodium levels in group 2 were more homogeneous. Histologic structures of heart tissue were regularly in group 2. Apoptosis were increased in control group compared to hepcidin treated group. Conclusion: These results suggest that hepcidin may have a protective effect on the heart for the ischemia-reperfusion injury.

Background and Aim: Momordica charantia is a highly valued plant, widely distributed in the tropical and subtropical regions. The plant is reported to have a wide range of medicinal uses. This study was designed to explore the ameliorative potential of M. charantia methanol leaf extract in alloxan-induced diabetic animal model with a particular focus on the liver. Materials and Methods: Hepatoprotective effect of methanol leaf extract of M. charantia was assessed in alloxan-induced toxicity in 50 rats divided into five groups (A-E) (n=10). Group A normal control, Group B was toxicant group, and Group C animals received glibenclamide treatment while Groups D and E received extracts at 200 and 400 mg/kg doses, respectively. The experiment lasted for 28 days. Histopathological changes, blood glucose level, and serum enzymes such as aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase, oxidative status and caspase-9, and interleukin-1β (IL-1β) were evaluated. Results: Extract-treatment caused a decreased blood glucose level, markers of oxidative stress such as malondialdehyde and hydrogen peroxide (H2O2). Treatment of rats with leaf extract of M. charantia resulted in increased levels and activities of protein thiols, non-protein thiols, glutathione (GSH), glutathione peroxidase, glutathione S-transferase, and superoxide dismutase indicating its antioxidant potential. The liver section revealed mild distortion of the hepatic architecture compared to the toxicant group, while decreased expressions of caspase-9 and IL-1β in extract-treated groups was observed. Conclusion: The plant extract exhibited antioxidant, anti-apoptotic, and anti-inflammatory effects, thus showing its hepatoprotective property.

Spinal cord injury (SCI) leads to abnormal expression of miRs, leading to secondary responses such as oxidative stress, inflammation and apoptosis. In the present work, we screened the miRs involved and the associated pathway.

Caspases are enzymes which are the main pathways for apoptosis. Any irregulation in their functions causes increase or decrease in cell death, which result in autoimmune disease or cancer, respectively. In this study, structure-based pharmacophore drug discovery method as a virtual screening was used to discover selective inhibitors for caspase-9. This enzyme is an initiator caspase that is the main pathway in Alzheimer’s disease. A pharmacophore model was developed by investigating essential interactions among the reported potent inhibitors employing a docking analysis methodology. Applying pharmacophore virtual screening, nine compounds from both National Cancer Institute (NCI), and ZINC databases were filtered as potent inhibitors of caspase-9. The efficiency of the discovered compounds was further investigated by docking studies. HIGHLIGHTS •Caspase-9 is an important enzyme for apoptosis and its activity is pivotal in cell death. •A computational design of small molecular inhibitors for caspase-9 performed by structure-based pharmacophore model. •9 compounds from both National Cancer Institute (NCI) and ZINC databases were discovered as potent inhibitors.

Abstract This study aimed to evaluate the specific regulatory roles of microRNA-146a (miRNA-146a) in temporal lobe epilepsy (TLE) and explore the related regulatory mechanisms. A rat model of TLE was established by intraperitoneal injection of lithium chloride-pilocarpine. These model rats were injected intracerebroventricularly with an miRNA-146a inhibitor and Notch-1 siRNA. Then, neuronal damage and cell apoptosis in the cornu ammonis (CA) 1 and 3 regions of the hippocampus were assessed. SOD and MDA levels in the hippocampus were detected by chromatometry, and IL-1β, IL-6, and IL-18 levels were detected by ELISA. Then, we evaluated the expression levels of caspase-9, GFAP, Notch-1, and Hes-1 in the hippocampus. The interaction between Notch-1 and miRNA-146a was assessed by a dual luciferase reporter gene assay. A rat model of TLE was successfully established, which exhibited significantly increased miRNA-146a expression in the hippocampus. Silencing of miRNA-146a significantly alleviated the neuronal damage and cell apoptosis in the CA1 and CA3 regions of the hippocampus in TLE rats and decreased MDA, IL-1β, IL-6, and IL-18 levels and increased SOD levels in the hippocampus of TLE rats. In addition, silencing of miRNA-146a significantly decreased the expression levels of caspase-9, GFAP, Notch-1, and Hes-1 in the hippocampus of TLE rats. Notch-1 was identified as a target of miRNA-146a and silencing of Notch-1 aggravated the neuronal damage in the CA1 and CA3 regions. Silencing of miRNA-146a alleviated the neuronal damage in the hippocampus of TLE rats by down-regulating Notch-1.