Activation Of Pro-apoptotic Caspase-3 Research Articles

Effect of Pyridoxine Derivative B6NO on Transcription Factor Nrf2 Activity and Cytotoxic Properties of Doxorubicin In Vitro.

The effect of a new pyridoxine derivative B6NO on doxorubicin cytotoxicity and Nrf2-dependent cellular processes in vitro was studied. Antioxidant B6NO enhances the cytotoxic effect of doxorubicin on tumor cells, which is associated with G2/M cell division arrest and an increase in activity of proapoptotic enzyme caspase-3. The antioxidant promotes intracellular accumulation and nuclear translocation of Nrf2 transcription factor in non-tumor and tumor cells. In non-tumor cells, B6NO increases the expression of antioxidant system proteins and reduces ROS generation in the presence of doxorubicin. In tumor cells, no activation of Nrf2-dependent processes occurs under the action of the antioxidant. Our findings demonstrate the prospect of further studies of pyridoxine derivatives as antioxidants to reduce adverse reactions during chemotherapy.

Complexation of fungal extracellular nucleic acids by host LL-37 peptide shapes neutrophil response to Candida albicans biofilm.

Candida albicans remains the predominant cause of fungal infections, where adhered microbial cells form biofilms - densely packed communities. The central feature of C. albicans biofilms is the production of an extracellular matrix (ECM) consisting of polymers and extracellular nucleic acids (eDNA, eRNA), which significantly impedes the infiltration of host cells. Neutrophils, as crucial players in the innate host defense, employ several mechanisms to eradicate the fungal infection, including NETosis, endocytosis, or the release of granules containing, among others, antimicrobial peptides (AMPs). The main representative of these is the positively charged peptide LL-37 formed from an inactive precursor (hCAP18). In addition to its antimicrobial functions, this peptide possesses a propensity to interact with negatively charged molecules, including nucleic acids. Our in vitro studies have demonstrated that LL-37 contacting with C. albicans nucleic acids, isolated from biofilm, are complexed by the peptide and its shorter derivatives, as confirmed by electrophoretic mobility shift assays. We indicated that the generation of the complexes induces discernible alterations in the neutrophil response to fungal nucleic acids compared to the effects of unconjugated molecules. Our analyses involving fluorescence microscopy, flow cytometry, and Western blotting revealed that stimulation of neutrophils with DNA:LL-37 or RNA:LL-37 complexes hamper the activation of pro-apoptotic caspases 3 and 7 and fosters increased activation of anti-apoptotic pathways mediated by the Mcl-1 protein. Furthermore, the formation of complexes elicits a dual effect on neutrophil immune response. Firstly, they facilitate increased nucleic acid uptake, as evidenced by microscopic observations, and enhance the pro-inflammatory response, promoting IL-8 production. Secondly, the complexes detection suppresses the production of reactive oxygen species and attenuates NETosis activation. In conclusion, these findings may imply that the neutrophil immune response shifts toward mobilizing the immune system as a whole, rather than inactivating the pathogen locally. Our findings shed new light on the intricate interplay between the constituents of the C. albicans biofilm and the host's immune response and indicate possible reasons for the elimination of NETosis from the arsenal of the neutrophil response during contact with the fungal biofilm.

Endothelial Progenitor Cells Affect the Growth and Apoptosis of Renal Cells by Secreting Microvesicles Carrying Dysregulated miR-205 and miR-206.

This study investigated the mechanism of microRNA (miRNA, miR) in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) involved in renal function in vivo and in vitro injury repair of rat primary kidney cells (PRKs). Gene Expression Omnibus analysis of potential target miRNAs in nephrotic rats. Real-time quantitative polymerase chain reaction verified the correlation of these miRNAs and screened the effective target miRNAs and their downstream putative target mRNAs. Western blot analyzes the protein levels of DEAD-box helicase 5 (DDX5) and the activation of the proapoptotic factor caspase-3/9 (cleaved). Dil-Ac-LDL staining, immunofluorescence, and a transmission electron microscope (TEM) were used to identify the successful isolation of EPCs and PRKs and the morphology of MVs. Cell Counting Kit-8 was used to detect the effect of miRNA-mRNA on the proliferation of PRKs. Standard biochemical kits were used to detect biochemical indicators in rat blood and urine. Dual-luciferase analysis of miRNA binding to mRNA was conducted. The effect of miRNA-mRNA interaction on the apoptosis level of PRKs was analyzed by flow cytometry. A total of 13 rat-derived miRNAs were potential therapeutic targets, and miR-205 and miR-206 were screened as the targets of this study. We found that the EPC-MVs alleviated the increase of blood urea nitrogen and urinary albumin excretion and the decrease in creatinine clearance caused by hypertensive nephropathy in vivo. The effect of MVs in improving renal function indicators was promoted by miR-205 and miR-206 and inhibited by knockdown of expressed miR-205 and miR-206. In vitro, angiotensin II (Ang II) promoted growth inhibition and apoptosis of PRKs, and similarly, dysregulated miR-205 and miR-206 affected the induction of Ang II. We then observed that miR-205 and miR-206 cotargeted the downstream target DDX5 and regulated its transcriptional activity and translational levels, while also reducing the activation of proapoptotic factors caspase-3/9. Overexpressed DDX5 reversed the effects of miR-205 and miR-206. By upregulating the expression of miR-205 and miR-206 in MVs secreted by EPC, the transcriptional activity of DDX5 and the activation of caspase-3/9 can be inhibited, thereby promoting the growth of PRKs and protecting the injury caused by hypertensive nephropathy.

Rift Valley Fever Virus Non-Structural Protein S Is Associated with Nuclear Translocation of Active Caspase-3 and Inclusion Body Formation.

Rift Valley fever phlebovirus (RVFV) causes Rift Valley fever (RVF), an emerging zoonotic disease that causes abortion storms and high mortality rates in young ruminants as well as severe or even lethal complications in a subset of human patients. This study investigates the pathomechanism of intranuclear inclusion body formation in severe RVF in a mouse model. Liver samples from immunocompetent mice infected with virulent RVFV 35/74, and immunodeficient knockout mice that lack interferon type I receptor expression and were infected with attenuated RVFV MP12 were compared to livers from uninfected controls using histopathology and immunohistochemistry for RVFV nucleoprotein, non-structural protein S (NSs) and pro-apoptotic active caspase-3. Histopathology of the livers showed virus-induced, severe hepatic necrosis in both mouse strains. However, immunohistochemistry and immunofluorescence revealed eosinophilic, comma-shaped, intranuclear inclusions and an intranuclear (co-)localization of RVFV NSs and active caspase-3 only in 35/74-infected immunocompetent mice, but not in MP12-infected immunodeficient mice. These results suggest that intranuclear accumulation of RVFV 35/74 NSs is involved in nuclear translocation of active caspase-3, and that nuclear NSs and active caspase-3 are involved in the formation of the light microscopically visible inclusion bodies.

Downregulating Mitochondrial DNA Polymerase γ in the Muscle Stimulated Autophagy, Apoptosis, and Muscle Aging-Related Phenotypes in Drosophila Adults.

Reactive oxygen species, generated as by-products of mitochondrial electron transport, can induce damage to mitochondrial DNA (mtDNA) and proteins. Here, we investigated whether the moderate accumulation of mtDNA damage in adult muscles resulted in accelerated aging-related phenotypes in Drosophila. DNA polymerase γ (Polγ) is the sole mitochondrial DNA polymerase. The muscle-specific silencing of the genes encoding the polymerase subunits resulted in the partial accumulation of mtDNA with oxidative damage and a reduction in the mtDNA copy number. This subsequently resulted in the production of abnormal mitochondria with reduced membrane potential and, consequently, a partially reduced ATP quantity in the adult muscle. Immunostaining indicated a moderate increase in autophagy and mitophagy in adults with RNA interference of Polγ (PolγRNAi) muscle cells with abnormal mitochondria. In adult muscles showing continuous silencing of Polγ, malformation of both myofibrils and mitochondria was frequently observed. This was associated with the partially enhanced activation of pro-apoptotic caspases in the muscle. Adults with muscle-specific PolγRNAi exhibited a shortened lifespan, accelerated age-dependent impairment of locomotor activity, and disturbed circadian rhythms. Our findings in this Drosophila model contribute to understanding how the accumulation of mtDNA damage results in impaired mitochondrial activity and how this contributes to muscle aging.

SOX9 Protein in Pancreatic Cancer Regulates Multiple Cellular Networks in a Cell-Specific Manner.

SOX9 is upregulated in the majority of pancreatic ductal adenocarcinoma cases. It is hypothesized that the increased expression of SOX9 is necessary for the formation and maintenance of tumor phenotypes in pancreatic cancer cells. In our research, we studied six pancreatic cancer cell lines, which displayed varying levels of differentiation and a range of oncogenic mutations. We chose the method of downregulation of SOX9 expression via siRNA transfection as the main method for investigating the functional role of the SOX9 factor in pancreatic cancer cells. We discovered that the downregulation of SOX9 expression in the cell lines leads to cell-line-specific changes in the expression levels of epithelial and mesenchymal protein markers. Additionally, the downregulation of SOX9 expression had a specific effect on the expression of pancreatic developmental master genes. SOX9 downregulation had the greatest effect on the expression levels of the protein regulators of cell proliferation. In three of the four cell lines studied, the transfection of siSOX9 led to a significant decrease in proliferative activity and to the activation of proapoptotic caspases in transfected cells. The acquired results demonstrate that the SOX9 protein exerts its multiple functions as a pleiotropic regulator of differentiation and a potential promoter of tumor growth in a cell-specific manner in pancreatic cancer cells.

Abstract 5315: Anti-tumor activity of KRASG12C inhibitors is enhanced when combined with Cdc42 effector p21-activated kinase 4 targeting agents

Abstract Background: KRASG12C inhibitors sotorasib and adagrasib have shown impressive results in clinical studies. These inhibitors efficiently block KRAS downstream effectors such as RAF, MEK, and ERK/p-ERK. However, factors such as feedback reactivation or bypass of KRAS dependence are emerging, resulting in the need for rational combination approaches. KRASG12C inhibitors have limited impact on the parallel oncogenic signaling driven by PI3K/AKT, its regulator molecules such as the Rho GTPases, and their effectors. This signaling pathway can collectively promote p-ERK re-activation thereby blunting the efficacy of KRASG12C targeted drugs. The p21-activated kinase (PAK) family are crucial effectors of the Rho family of GTPases and act as regulatory switches that control important cellular processes including motility, proliferation, and survival. PAK4 is the main effector of Rho GTPase, cell division control protein 42 homolog (Cdc42), and has an established role in mutant RAS-dependent tumor metastasis. In this study, we rationally targeted two parallel signaling pathways by combining KRASG12C inhibitors with the PAK4 inhibitor KPT-9274 in KRASG12C mutant PDAC and NSCLC in vitro and in vivo models. Methods: We evaluated the cytotoxicity and molecular profile of KRASG12C inhibitors in combination with PAK4 inhibitor KPT-9274 as well as positive control PF-3758309 in KRASG12C mutant 2D and 3D cellular/spheroid models. The anti-tumor activity of the combinations was evaluated in KRASG12C mutant cell line-derived xenograft. Tumor volumes were assessed throughout the treatment period using an ultrasound scanner. Results: PAK4 inhibitor KPT-9274 synergized with both sotorasib and adagrasib yielding suppressed growth of KRASG12C mutant PDAC and NSCLC cells in 2D and 3D cultures, but not in KRASwt, KRASG12V, and KRASG12D cell lines (NCI RASless MEFs). In addition, the combination reduced the clonogenic potential of KRASG12C mutant cancer cells. PAK4-KRASG12C inhibitor combinations resulted in enhanced and prolonged inhibition of KRAS downstream effector pathways. In xenograft studies, oral administration of KPT-9274 at sub-optimal dose (100 mg/kg QD x 5 x 3 weeks) and sotorasib at one-fourth of MTD (25 mg/kg QD x 5 x 3 weeks) demonstrated remarkable inhibition of KRASG12C mutant MiaPaCa-2 tumor burden (p<0.001 single agents vs combination treatment). Residual tumor analysis showed a reduction in proliferation index (ki67), activation of pro-apoptotic caspases, and inhibition of mutant RAS effector signaling. Studies in additional KRASG12C xenograft and tumor explant models are ongoing. Conclusion: This is the first study showing that KRASG12C inhibitors can synergize with PAK4 inhibitors. This combination can simultaneously target direct KRAS downstream effectors and the parallel PI3K/AKT oncogenic signaling warranting further clinical investigations. Citation Format: Husain Yar Khan, Sahar Bannoura, Hirva Mamdani, Amro Aboukameel, Yousef Mzannar, Yiwei Li, Mohammad Najeeb Al-Hallak, Ibrahim Azar, Steve Kim, Rafic Beydoun, Ramzi Mohammad, Yosef Landesman, Yue Zhang, Erkan Baloglu, William Senapedis, Ammar Sukari, Misako Nagasaka, Anthony Shields, Asfar S. Azmi. Anti-tumor activity of KRASG12C inhibitors is enhanced when combined with Cdc42 effector p21-activated kinase 4 targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5315.

Vitamin Supplementation Protects against Nanomaterial-Induced Oxidative Stress and Inflammation Damages: A Meta-Analysis of In Vitro and In Vivo Studies

The extensive applications of nanomaterials have increased their toxicities to human health. As a commonly recommended health care product, vitamins have been reported to exert protective roles against nanomaterial-induced oxidative stress and inflammatory responses. However, there have been some controversial conclusions in regards to this field of research. This meta-analysis aimed to comprehensively evaluate the roles and mechanisms of vitamins for cells and animals exposed to nanomaterials. Nineteen studies (seven in vitro, eleven in vivo and one in both) were enrolled by searching PubMed, EMBASE, and Cochrane Library databases. STATA 15.0 software analysis showed vitamin E treatment could significantly decrease the levels of oxidants [reactive oxygen species (ROS), total oxidant status (TOS), malondialdehyde (MDA)], increase anti-oxidant glutathione peroxidase (GPx), suppress inflammatory mediators (tumor necrosis factor-α, interleukin-6, C-reactive protein, IgE), improve cytotoxicity (manifested by an increase in cell viability and a decrease in pro-apoptotic caspase-3 activity), and genotoxicity (represented by a reduction in the tail length). These results were less changed after subgroup analyses. Pooled analysis of in vitro studies indicated vitamin C increased cell viability and decreased ROS levels, but its anti-oxidant potential was not observed in the meta-analysis of in vivo studies. Vitamin A could decrease MDA, TOS and increase GPx, but its effects on these indicators were weaker than vitamin E. Also, the combination of vitamin A with vitamin E did not provide greater anti-oxidant effects than vitamin E alone. In summary, we suggest vitamin E alone supplementation may be a cost-effective option to prevent nanomaterial-induced injuries.

Effect of miltirone on proliferation and apoptosis of leukemia THP-1 cells

To investigate the toxicity and related mechanism of miltirone to human acute myeloid leukemia THP-1 cells. To be specific, the active components and targets of miltirone were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP), and the target proteins were converted into standard gene names with UniProt. Acute leukemia-rela-ted target genes were screened from GeneCards and DisGeNET. Venn diagram was constructed with Venny 2.1 to yield the common targets of the disease and the drug. The protein-protein interaction(PPI) network was constructed by STRING and Cytoscape 3.8.2. THP-1 cells in the logarithmic growth phase were treated with dimethyl sulfoxide(DMSO), and 2.5, 5, 10, 15, and 20 μmol·L~(-1) miltirone for 24 h, respectively. The proliferation rate of cells was analyzed by carboxyfluorescein diacetate succinimidyl ester(CFSE), apoptosis rate by flow cytometry with Annexin V-PE/7 AAD staining, and cell morphology by acridine orange staining. Real-time quantitative PCR(qPCR) was employed to detect the mRNA levels of nuclear receptor coactivator 2(NCOA2), poly(ADP-ribose) polymerase-1(PARP1), B-cell lymphoma-2(Bcl-2)-associated X protein(Bax), Bcl-2, and cysteine aspartyl protease-3(caspase-3). The effect of miltirone on apoptosis was detected in presence of caspase inhibitor Z-VAD-FMK. A total of 26 targets of miltirone, 1 046 genes related to acute leukemia, and 6 common targets of the two were screened out. Flow cytometry result showed miltirone at 10 μmol·L~(-1) can inhibit proliferation and promote apoptosis of THP-1 cells. The typical manifestations of apoptosis, such as cell shrinkage, nuclear rupture, and chromatin agglomerate were displayed by acridine orange staining. The decreased mRNA levels of NCOA2 and PARP1 and increased Bax/Bcl-2 ratio and the activity of pro-apoptotic protein caspase-3 were observed. Z-VAD-FMK can attenuate the apoptosis-inducing effect of miltirone. This study indicates that miltirone can inhibit the proliferation and promote the apoptosis of THP-1 cells, by down-regulating NCOA2 and PARP1, raising Bax/Bcl-2 ratio, and activating caspase-3.

Neurogenic and cognitive enhancing effects of human dental pulp stem cells and its secretome in animal model of hippocampal neurodegeneration

Progressive hippocampal neuronal losses, neuroinflammation, declined neurogenesis and impaired hippocampal functions are pathological features of Alzheimer’s disease and temporal lobe epilepsy (TLE). Halting neuroinflammation and progressive neurodegeneration in the hippocampus is a major challenge in treating such disease conditions which, if unsuccessful would lead to learning/memory dysfunction and co-morbidities like anxiety/depression. Mesenchymal stem cells (MSCs) therapy provides hope for treating neurodegenerative diseases by either replacing lost neurons by transplantation of MSCs which might differentiate into appropriate neuronal phenotypes or by stimulating the resident neural stem cells for proliferation/differentiation. In this current study, we demonstrate that the intrahippocampal transplantation of ectoderm originated dental pulp stem cells (DPSCs) or intrahippocampal injection of DPSCs condition medium (DPSCs-CM) in a mouse model of hippocampal neurodegeneration could efficiently prevent neurodegeneration, neuroinflammation, enhance hippocampal neurogenesis and spatial learning and memory functions much superior to commonly used bone marrow mesenchymal stem cells (BM-MSCs) or its secretome. Probing the possible mechanisms of neuroprotection revealed that DPSCs/DPSCs-CM treatment upregulated an array of hosts’ endogenous neural survival factors expression, reduced pro-apoptotic caspase activity and upregulated the anti-apoptotic factors BCL-2 and phosphorylated PI3K prominently than BM-MSCs/BM-MSCs-CM, suggesting that among MSCs, neural crest originated DPSCs might be a better adult stem cell candidate for treating neurodegenerative diseases.

Lycium barbarum polysaccharides and ferroptosis: jumping into the era of novel regulated cell death.

Lycium barbarum polysaccharides and ferroptosis: jumping into the era of novel regulated cell death.

ZNF598 co-translationally titrates poly(GR) protein implicated in the pathogenesis of C9ORF72-associated ALS/FTD.

C9ORF72-derived dipeptide repeat proteins have emerged as the pathogenic cause of neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). However, the mechanisms underlying their expression are not fully understood. Here, we demonstrate that ZNF598, the rate-limiting factor for ribosome-associated quality control (RQC), co-translationally titrates the expression of C9ORF72-derived poly(GR) protein. A Drosophila genetic screen identified key RQC factors as potent modifiers of poly(GR)-induced neurodegeneration. ZNF598 overexpression in human neuroblastoma cells inhibited the nuclear accumulation of poly(GR) protein and decreased its cytotoxicity, whereas ZNF598 deletion had opposing effects. Poly(GR)-encoding sequences in the reporter RNAs caused translational stalling and generated ribosome-associated translation products, sharing molecular signatures with canonical RQC substrates. Furthermore, ZNF598 and listerin 1, the RQC E3 ubiquitin-protein ligase, promoted poly(GR) degradation via the ubiquitin-proteasome pathway. An ALS-relevant ZNF598R69C mutant displayed loss-of-function effects on poly(GR) expression, as well as on general RQC. Moreover, RQC function was impaired in C9-ALS patient-derived neurons, whereas lentiviral overexpression of ZNF598 lowered their poly(GR) expression and suppressed proapoptotic caspase-3 activation. Taken together, we propose that an adaptive nature of the RQC-relevant ZNF598 activity allows the co-translational surveillance to cope with the atypical expression of pathogenic poly(GR) protein, thereby acquiring a neuroprotective function in C9-ALS/FTD.

IL-1R1 deficiency impairs liver regeneration after 2/3 partial hepatectomy in aged mice.

Inflammation has a dual effect: it can protect the body and destroy tissue and cell as well. The purpose of this experiment was to determine the role of IL-1R1 in liver regeneration (LR) after partial hepatectomy (PH) in aged mice. The wild-type (WT, n = 36) and the IL-1R1 knockout (KO, n = 36) 24-month-old C57BL/6J mice underwent two-thirds PH; 33 WT mice underwent sham operation. Liver coefficient was calculated by liver/body weight. The mRNA and protein expressions of genes were evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting methods, respectively. Compared with WT mice, liver coefficient was lower in the IL-1R1 KO aged mice at 168 and 192 h (p = 0.039 and p = 0.027). The mRNA transcription of inflammation-related genes and cell cycle-associated genes decreased or delayed. The protein expressions of proliferation-related marker PCNA and proliferation-associated signaling pathway components JNK1, NF-κB and STAT3 reduced or retarded. There was stronger activation of proapoptotic proteins caspase-3, caspase-8 and BAX in the IL-1R1 KO mice at different time points (p < 0.05 or p < 0.01). IL-1R1 KO reduced inflammation and caused impaired liver regeneration after 2/3 partial hepatectomy in aged mice. Maintaining proper inflammation may contribute to regeneration after liver partly surgical resection in the elderly.

Effects of prenatal synthetic cannabinoid exposure on the cerebellum of adolescent rat offspring

Cannabis is the most commonly used illicit drug worldwide. Recently, cannabis use among young pregnant women has greatly increased. However, prenatal cannabinoid exposure leads to long-lasting cognitive, motor, and behavioral deficits in the offspring and alterations in neural circuitry through various mechanisms. Although these effects have been studied in the hippocampus, the effects of prenatal cannabinoid exposure on the cerebellum are not well elucidated. The cerebellum plays an important role in balance and motor control, as well as cognitive functions such as attention, language, and procedural memories. The aim of this study was to investigate the effects of prenatal cannabinoid exposure on the cerebellum of adolescent offspring. Pregnant rats were treated with synthetic cannabinoid agonist WIN55,212-2, and the offspring were evaluated for various cerebellar markers of oxidative stress, mitochondrial function, and apoptosis. Additionally, signaling proteins associated with glutamate dependent synaptic plasticity were examined. Administration of WIN55,212-2 during pregnancy altered markers of oxidative stress by significantly reducing oxidative stress and nitrite content. Mitochondrial Complex I and Complex IV activities were also enhanced following prenatal cannabinoid exposure. With regard to apoptosis, pP38 levels were significantly increased, and proapoptotic factor caspase-3 activity, pERK, and pJNK levels were significantly decreased. CB1R and GluA1 levels remained unchanged; however, GluN2A was significantly reduced. There was a significant decrease in MAO activity although tyrosine hydroxylase activity was unaltered. Our study indicates that the effects of prenatal cannabinoid exposure on the cerebellum are unique compared to other brain regions by enhancing mitochondrial function and promoting neuronal survival. Further studies are required to evaluate the mechanisms by which prenatal cannabinoid exposure alters cerebellar processes and the impact of these alterations on behavior.

Schistosoma mansoni alter transcription of immunomodulatory gene products following in vivo praziquantel exposure

Control of the neglected tropical disease schistosomiasis relies almost entirely on praziquantel (PZQ) monotherapy. How PZQ clears parasite infections remains poorly understood. Many studies have examined the effects of PZQ on worms cultured in vitro, observing outcomes such as muscle contraction. However, conditions worms are exposed to in vivo may vary considerably from in vitro experiments given the short half-life of PZQ and the importance of host immune system engagement for drug efficacy in animal models. Here, we investigated the effects of in vivo PZQ exposure on Schistosoma mansoni. Measurement of pro-apoptotic caspase activation revealed that worm death occurs only after parasites shift from the mesenteric vasculature to the liver, peaking 24 hours after drug treatment. This indicates that PZQ is not directly schistocidal, since PZQ’s half-life is ~2 hours in humans and ~30 minutes in mice, and focuses attention on parasite interactions with the host immune system following the shift of worms to the liver. RNA-Seq of worms harvested from mouse livers following sub-lethal PZQ treatment revealed drug-evoked changes in the expression of putative immunomodulatory and anticoagulant gene products. Several of these gene products localized to the schistosome esophagus and may be secreted into the host circulation. These include several Kunitz-type protease inhibitors, which are also found in the secretomes of other blood feeding animals. These transcriptional changes may reflect mechanisms of parasite immune-evasion in response to chemotherapy, given the role of complement-mediated attack and the host innate/humoral immune response in parasite elimination. One of these isoforms, SmKI-1, has been shown to exhibit immunomodulatory and anti-coagulant properties. These data provide insight into the effect of in vivo PZQ exposure on S. mansoni, and the transcriptional response of parasites to the stress of chemotherapy.

MicroRNA-365 promotes apoptosis in human melanoma cell A375 treated with hydatid cyst fluid of Echinococcus granulosus sensu stricto

Hydatid cyst fluid (HCF)-based therapeutics has experimentally targeted approaches for treating human cancer cell lines. MicroRNA-365 (miR-365) has been reported to be an important tumor suppressor miRNA in cancers. However, it remains unknown, how miR-365 plays a pivotal role in inducing apoptosis in HCF-treated cancer cells in vitro. The fertile/infertile HCF was aspirated from liver of infected sheep and in terms of molecular taxonomy was identified as G1 genotype of Echinococcus granulosus sensu stricto. A375 human melanoma cancer cells were cultured into two groups: fertile and infertile HCF-treated A375 cells. To assess the cytotoxicity of various concentrations of HCF on melanoma cells, cell viability was determined by using MTT assay. The IC50 value of HCF on A375 cells was determined 85 μg/mL. Caspase-3 enzymatic activity was evaluated by fluorometric assay in the HCF-treated melanoma cells. In addition, the mRNA expression of Bax, Bcl-2, Caspase-9 and miR-365 were determined by qRT-PCR. Findings of MTT assay showed that concentrations 85 μg/mL to 100 μg/mL of fertile HCF have the highest mortality (50%–52%) on A375 cells during 24 h. The fold change of Bax/Bcl-2 ratio, Caspase-9, miR-365 and Caspase-3 activity was higher in the fertile HCF-treated melanoma cells compared to infertile fluid treated A375 cells and human normal epithelial cell (as control cell). In conclusion, we over-expressed the miR-365 in melanoma A375 cells, via treatment of fertile HCF. Our findings suggested that inducing high expression of miR-365 might be a negative regulator of melanoma growth through activation of pro-apoptotic Bax, Caspase-9 and Caspase-3 that are essential to intrinsic apoptotic pathway. These findings provide new insights into the use of Echinococcus HCF-derived metabolites in the design of drug therapies and in vivo tumor cell vaccine to combat melanoma progression.

QbD Enabled Azacitidine Loaded Liposomal Nanoformulation and Its In Vitro Evaluation.

Azacitidine (AZA), an inhibitor of DNA methyltransferase, is a commonly recognized drug used in clinical treatment for myelodysplastic syndrome and breast cancer. Due to higher aqueous solubility and negative log P of AZA causes poor cancer cell permeation and controlled release. The objective of the present study was to formulate and optimize AZA-loaded liposome (AZA-LIPO) for breast cancer chemotherapy by using Box Behnken design (BBD) and in vitro evaluation using MCF-7 cells. AZA-LIPO were prepared using a thin film hydration technique and characterization study was performed by using FTIR and DSC. The prepared formulations were optimized using BBD and the optimized formulation was further subjected for particle size, surface charges, polydispersity index (PDI), drug loading, entrapment efficiency, TEM, XRD, in-vitro drug release and hemolytic toxicity. The mean particle size of optimized AZA-LIPO was 127 nm. Entrapment efficiency and drug loading of AZA-LIPO was found to be 85.2% ± 0.5 and 6.82 ± 1.6%, respectively. Further, in vitro drug release study showed preliminary burst release in 2 h followed by a sustained release for 36 h in phosphate buffer at different pH (4.0, 5.5, and 7.4) as compared to free drug. Drug release was found to be pH dependent, as the pH was increased, the drug release rate was found to be low. Time-dependent cell viability assay exhibited significant higher cell viability and higher internalization than free AZA in MCF-7 cells. AZA-LIPO were more effective than the free AZA in reducing Bcl2 expression, while increasing pro-apoptotic Bax and caspase-3 activity. The result showed that the formulated biocompatible AZA-LIPO nano-formulations may be used as an efficient anti-cancer drug delivery system for the treatment of breast cancer after establishing preclinical and clinical studies.

The Anti-Cancer Effect of Linusorb B3 from Flaxseed Oil through the Promotion of Apoptosis, Inhibition of Actin Polymerization, and Suppression of Src Activity in Glioblastoma Cells.

Linusorbs (LOs) are natural peptides found in flaxseed oil that exert various biological activities. Of LOs, LOB3 ([1–9-NαC]-linusorb B3) was reported to have antioxidative and anti-inflammatory activities; however, its anti-cancer activity has been poorly understood. Therefore, this study investigated the anti-cancer effect of LOB3 and its underlying mechanism in glioblastoma cells. LOB3 induced apoptosis and suppressed the proliferation of C6 cells by inhibiting the expression of anti-apoptotic genes, B cell lymphoma 2 (Bcl-2) and p53, as well as promoting the activation of pro-apoptotic caspases, caspase-3 and -9. LOB3 also retarded the migration of C6 cells, which was achieved by suppressing the formation of the actin cytoskeleton critical for the progression, invasion, and metastasis of cancer. Moreover, LOB3 inhibited the activation of the proto-oncogene, Src, and the downstream effector, signal transducer and activator of transcription 3 (STAT3), in C6 cells. Taken together, these results suggest that LOB3 plays an anti-cancer role by inducing apoptosis and inhibiting the migration of C6 cells through the regulation of apoptosis-related molecules, actin polymerization, and proto-oncogenes.

Pterostilbene as a Phytochemical Compound Induces Signaling Pathways Involved in the Apoptosis and Death of Mutant P53-Breast Cancer Cell Lines

Pterostilbene is a natural nonflavonoid polyphenolic compound. It shows a remarkable range of biological activities, including antiproliferative, antiinflammatory, and antioxidant activity. However, the mechanism of action of PT in breast cancer cells containing mutant p53 protein has not been fully elucidated. Therefore, the present study was aimed at investigating the influence of PT on signaling pathways involved in the apoptosis of mutant p53-breast cancer cell lines. Immunocytochemistry and Western Immunoblotting techniques were used in this study. The present data showed that the viabilities and the proliferations of MDA-MB-231 and T-47D decreased significantly (P < 0.001) after treatment with different concentrations of PT. In addition, the morphological characteristics of both cell lines were changed after treatment with PT. Decreased protein expression of mutant p53 (R280 K, L194F) in MDA-MB-231 and T-47D breast cancer cell lines has also been achieved. In addition, overexpression of pro-apoptotic (Bax) protein, caspase-3 activity and histone release were increased after treatment of both cell lines with different PT concentrations. Furthermore, the protein expressions of cyclin D1, mTOR, and oncogenic β-catenin were significantly downregulated after treatment of both cell lines with PT. In conclusion, downregulations of protein expression of mutant p53, cyclin D1, mTOR, and β-catenin were increased after both cell lines had been treated with pterostilbene. PT could point to a promising use against the development and the progression of breast cancer as a natural therapeutic agent.

Formulation Design, Statistical Optimization, and In Vitro Evaluation of a Naringenin Nanoemulsion to Enhance Apoptotic Activity in A549 Lung Cancer Cells.

Naringenin (NAR), a flavonoid mainly found in citrus and grapefruits, has proven anti-cancer activities. However, the poor water solubility and low bioavailability of NAR limits its use as a therapeutic agent. The aim of this study was to develop and optimize stable naringenin nanoemulsions (NAR-NE) using a Box–Behnken experimental design to obtain a formulation with a higher efficiency. Anticancer activity of optimized NAR-NE was evaluated in A549 lung cancer cells using cell viability, flow-cytometric assays, and enzyme-linked immunosorbent assay. The stabilized nanoemulsion, which showed a spherical surface morphology, had a globule size of 85.6 ± 2.1 nm, a polydispersity index of 0.263 ± 0.02, a zeta potential of −9.6 ± 1.2 mV, and a drug content of 97.34 ± 1.3%. The NAR release from the nanoemulsion showed an initial burst release followed by a stable and controlled release for a longer period of 24 h. The nanoemulsion exhibited excellent thermodynamic and physical stability against phase separation and storage. The NAR-NE showed concentration-dependent cytotoxicity in A549 lung cancer cells, which was greater than that of free NAR. The percentage of apoptotic cells and cell cycle arrest at the G2/M and pre-G1 phases induced by NAR-NE were significantly higher than those produced by free NAR (p < 0.05). NAR-NEs were more effective than the NAR solution in reducing Bcl2 expression, while increasing pro-apoptotic Bax and caspase-3 activity. Therefore, stabilized NAR-NE could be a suitable drug delivery system to enhance the effects of NAR in the treatment of lung cancer.