ABSTRACT
Dengue virus (DENV) is a major global health concern, causing millions of infections annually. Understanding the cellular response to DENV infection is crucial for developing effective therapies. This study provides an in-depth analysis of the cellular response to Dengue virus (DENV) infection, with a specific focus on the interplay between microRNAs (miRNAs), apoptosis, and viral load across different DENV serotypes. Utilizing a variety of cell lines infected with four DENV serotypes, the research methodically quantifies viral load, and the expression levels of miRNA-15, miRNA-16, and BCL2 protein, alongside measuring apoptosis markers. Methodologically, the study employs quantitative PCR for viral load and miRNA expression analysis, and Western blot for apoptosis and BCL2 detection, with a statistical framework that includes ANOVA and correlation analysis to discern significant differences and relationships. The findings reveal that despite similar viral loads across DENV serotypes, DENV-2 exhibits a marginally higher load. A notable upregulation of miRNA-15 and miRNA-16 correlates positively with increased viral load, suggesting their potential role in modulating viral replication. Concurrently, a marked activation of caspases 3 and 7, along with changes in BCL2 protein levels, underscores the role of apoptosis in the cellular response to DENV infection. Conclusively, the study enhances the understanding of miRNA involvement in DENV pathogenesis, highlighting miRNA-15 and miRNA-16 as potential regulatory agents in viral replication and apoptosis. These findings pave the way for further exploration into miRNA-based therapeutic strategies against DENV infection.
Subject(s)
Apoptosis , Dengue Virus , Dengue , MicroRNAs , Proto-Oncogene Proteins c-bcl-2 , Viral Load , Virus Replication , MicroRNAs/genetics , MicroRNAs/metabolism , Dengue Virus/physiology , Dengue Virus/genetics , Humans , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Dengue/virology , Cell Line , Caspase 3/metabolism , Caspase 3/genetics , Caspase 7/metabolism , Caspase 7/genetics , SerogroupABSTRACT
OBJECTIVE: Bisphenol F (BPF) is an endocrinedisrupting chemical, but information about its effect on thyroid hormones has not been fully explored. Omega 3 fatty acids (O3FA), on the other hand, are antioxidant and antiapoptotic agents. Therefore, this study explored the role and associated molecular mechanism of O3FA in BPF-induced hypothyroidism-mediated testicular dysfunction in male Wistar rats. METHODS: Twenty (20) male Wistar rats were randomized into four groups (n=5/group), namely: the control group; the BPF treated group (30 mg/kg of BPF); and the intervention groups (30mg/kg BPF + 100mg/kg O3FA (BPF+O3FA-L) and 30mg/kg BPF + 300mg/kg of O3FA for 28 days). RESULTS: Low and high doses of O3FA ameliorated BPF-induced hypothyroidism-mediated reduction in sperm quality, testosterone, luteinizing hormone, follicle-stimulating hormone, catalase, superoxide dismutase, total antioxidant capacity, and nuclear factor erythroid 2-related factor 2 and increases in estrogen, malondialdehyde, c-reactive protein, interleukin 1 beta, caspase 3. Furthermore, O3FA prevented BPF-induced Na+/K+-ATPase and Ca2+-ATPase dysfunction, estrogen receptor beta overexpression, and tumor protein P53 (p53)/ b-cell lymphoma 2 (Bcl-2) imbalance. CONCLUSIONS: This study showed that O3FA ameliorated BPF-induced dysthyroidism-mediated testicular dysfunction by preventing proton pump dysfunction and p53/BCl-2 imbalance.
Subject(s)
Fatty Acids, Omega-3 , Testis , Tumor Suppressor Protein p53 , Animals , Male , Rats , Fatty Acids, Omega-3/pharmacology , Hypothyroidism/metabolism , Hypothyroidism/chemically induced , Hypothyroidism/drug therapy , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats, Wistar , Signal Transduction/drug effects , Testis/drug effects , Testis/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Ilama leaves are an important source of secondary metabolites with promising anticancer properties. Cancer is a disease that affects a great number of people worldwide. This work aimed to investigate the in vivo, in vitro and in silico anticancer properties of three acyclic terpenoids (geranylgeraniol, phytol and farnesyl acetate) isolated from petroleum ether extract of ilama leaves. Their cytotoxic activity against U-937 cells was assessed using flow cytometry to determine the type of cell death and production of reactive oxygen species (ROS). Also, a morphological analysis of the lymph nodes and a molecular docking study using three proteins related with cancer as targets, namely, Bcl-2, Mcl-1 and VEGFR-2, were performed. The flow cytometry and histomorphological analysis revealed that geranylgeraniol, phytol and farnesyl acetate induced the death of U-937 cells by late apoptosis and necrosis. Geranylgeraniol and phytol induced a significant increase in ROS production. The molecular docking studies showed that geranylgeraniol had more affinity for Bcl-2 and VEGFR-2. In the case of farnesyl acetate, it showed the best affinity for Mcl-1. This study provides information that supports the anticancer potential of geranylgeraniol, phytol and farnesyl acetate as compounds for the treatment of cancer, particularly with the potential to treat non-Hodgkin's lymphoma.
Subject(s)
Molecular Docking Simulation , Plant Extracts , Plant Leaves , Plants, Medicinal , Reactive Oxygen Species , Humans , Plant Leaves/chemistry , Plants, Medicinal/chemistry , Plant Extracts/chemistry , Plant Extracts/pharmacology , Reactive Oxygen Species/metabolism , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/chemistry , Mexico , Apoptosis/drug effects , Cell Line, Tumor , Animals , Vascular Endothelial Growth Factor Receptor-2/metabolism , Vascular Endothelial Growth Factor Receptor-2/antagonists & inhibitors , Computer Simulation , Proto-Oncogene Proteins c-bcl-2/metabolism , U937 CellsABSTRACT
OBJECTIVE: To analyze the immunohistochemical expression of YAP and its correlation with markers involved in cell proliferation and apoptosis in benign epithelial odontogenic lesions. STUDY DESIGN: The sample consisted of 95 cases of odontogenic lesions (25 dentigerous cysts, 30 non-syndromic odontogenic keratocysts, 30 conventional ameloblastomas, and 10 unicystic ameloblastomas) and 10 dental follicles used as normal odontogenic tissue. The histological sections were submitted to immunohistochemistry with YAP, cyclin D1, Ki-67, and Bcl-2 antibodies. Immunoexpression was analyzed qualitatively and quantitatively using an adapted method. The collected data were analyzed descriptively and statistically (p ≤ 0.05). RESULTS: The highest YAP expression was observed in odontogenic keratocysts, followed by unicystic ameloblastomas and conventional ameloblastomas, which exhibited moderate immunoreactivity predominantly in peripheral cells. Furthermore, significant differences in YAP immunoexpression were observed between the groups analyzed, with significant positive correlations between YAP and cyclin D1 in dentigerous cysts and unicystic ameloblastomas and between YAP and Ki-67 in unicystic ameloblastomas (p < 0.05). However, there were no statistically significant correlations between YAP and Bcl-2 immunoexpression in the groups studied. CONCLUSION: YAP may influence epithelial cell proliferation in odontogenic cysts and tumors, suggesting its possible participation in the progression of the odontogenic lesions studied.
Subject(s)
Adaptor Proteins, Signal Transducing , Ameloblastoma , Apoptosis , Cell Proliferation , Cyclin D1 , Dentigerous Cyst , Ki-67 Antigen , Odontogenic Cysts , Proto-Oncogene Proteins c-bcl-2 , YAP-Signaling Proteins , Humans , Ameloblastoma/pathology , Ameloblastoma/metabolism , Odontogenic Cysts/pathology , Odontogenic Cysts/metabolism , Dentigerous Cyst/pathology , Dentigerous Cyst/metabolism , Ki-67 Antigen/metabolism , Ki-67 Antigen/analysis , Cyclin D1/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Proto-Oncogene Proteins c-bcl-2/analysis , Proto-Oncogene Proteins c-bcl-2/metabolism , Transcription Factors/analysis , Dental Sac/pathology , Dental Sac/metabolism , Immunohistochemistry , Odontogenic Tumors/pathology , Odontogenic Tumors/metabolism , Epithelial Cells/pathology , Epithelial Cells/metabolismABSTRACT
Phosphatidylinositol-5-phosphate 4-kinase type 2 (PIP4K2) protein family members (PIP4K2A, PIP4K2B, and PIP4K2C) participate in the generation of PIP4,5P2, which acts as a secondary messenger in signal transduction, a substrate for metabolic processes, and has structural functions. In patients with acute myeloid leukemia (AML), high PIP4K2A and PIP4K2C levels are independent markers of a worse prognosis. Recently, our research group reported that THZ-P1-2 (PIP4K2 pan-inhibitor) exhibits anti-leukemic activity by disrupting mitochondrial homeostasis and autophagy in AML models. In the present study, we characterized the expression of PIP4K2 in the myeloid compartment of hematopoietic cells, as well as in AML cell lines and clinical samples with different genetic abnormalities. In ex vivo assays, PIP4K2 expression levels were related to sensitivity and resistance to several antileukemia drugs and highlighted the association between high PIP4K2A levels and resistance to venetoclax. The combination of THZ-P1-2 and venetoclax showed potentiating effects in reducing viability and inducing apoptosis in AML cells. A combined treatment differentially modulated multiple genes, including TAp73, BCL2, MCL1, and BCL2A1. In summary, our study identified the correlation between the expression of PIP4K2 and the response to antineoplastic agents in ex vivo assays in AML and exposed vulnerabilities that may be exploited in combined therapies, which could result in better therapeutic responses.
Subject(s)
Antineoplastic Agents , Leukemia, Myeloid, Acute , Humans , Cell Line, Tumor , Proto-Oncogene Proteins c-bcl-2/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Apoptosis , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Phosphotransferases (Alcohol Group Acceptor)/pharmacologyABSTRACT
PURPOSE: Our aim was to investigate protective effects of daidzein treatment on ischemia-reperfusion (I/R) injury-induced ovarian tissue by immunohistochemical techniques. METHODS: Thirty Sprague Dawley female rats were categorized into three groups as sham, I/R group, and I/R+daidzein groups. Bloods were analyzed for malondialdehyde (MDA), glutathione peroxidase (GSH), and myeloperoxidase (MPO), and ovaries were processed for histological tissue protocol. RESULTS: Both MDA and MPO values were increased in I/R group compared to sham and I/R+daidzein groups. GSH content was increased in I/R+daidzein group compared to I/R groups. In I/R group, theca and follicular cells were degenerated with apoptosis and dilatation and congestion, edema. In I/R+daidzein group, daidzein improved pathologies. In the I/R group, Bax expression was positive with follicular cells, granulosa cells and inflammatory cells. In the I/R+daidzein group, positive Bax reaction was observed in the epithelial, antral, and inflammatory cells. In I/R group, Bcl-2 reaction was in germinative epithelial cells, cells of antral follicle. In the I/R+daidzein group, Bcl-2 expression level was reduced after daidzein treatment. CONCLUSIONS: After the I/R procedure, ovarian cells and follicles were degenerated with apoptosis and inflammation. After daidzein treatment, Bax and Bcl-2 signal were decreased. It was observed that daidzein stopped the apoptotic process.
Subject(s)
Ovary , Reperfusion Injury , Rats , Animals , Female , Rats, Sprague-Dawley , Ovary/pathology , bcl-2-Associated X Protein/metabolism , Ischemia/pathology , Reperfusion Injury/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Reperfusion , Malondialdehyde/metabolism , ApoptosisABSTRACT
This study evaluated the effects of laser application of diverse wavelengths applied simultaneously and on different skins. The sample included two participants, a woman with light skin with abdominal hair and a woman with dark skin and hair on the inner part of the lower limbs, who received a laser therapy session. After 45 days from laser application, abdominoplasty and thigh dermolipectomy surgery were performed. In the control sample, the hair follicles were in the anagen phase, showing the presence of Bcl-2 expression. In the treated areas, follicles were observed in an advanced phase (telogen), with the presence of CK-18 and negativity of Bcl-2, highlighting the phase of hair loss at that moment and the complete apoptosis of the investigated follicle. Significant difference was observed in the comparison of the anagen phase (p = .00) and it similarly occurred in the comparison of the telogen phase (p = .00). The presence of a greater amount of follicles in the anagen phase in the control area and follicles in the telogen phase in the treated area demonstrates the efficiency of the laser at different wavelengths when reaching different skin phototypes and hair thickness, being reinforced by apoptosis and cell proliferation markers. Therefore, the hair-removal process has been optimized with various laser wavelengths.
Subject(s)
Hair Removal , Female , Humans , Hair , Skin , Hair Follicle , Lasers , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
The present study aims to investigate if Cimicifuga racemosa (L.) Nutt extract (CIMI) reduces deleterious effects of dexamethasone (DEXA) in ovaries cultured in vitro. Mouse ovaries were collected and cultured in DMEM+ only or supplemented with 5 ng/mL of CIMI, or 4 ng/mL DEXA, or both CIMI and DEXA. The ovaries were cultured at 37.5°C in 5% CO2 for 6 days. Ovarian morphology, follicular ultrastructure, and the levels of mRNA for Bax, Bcl-2, and Caspase-3 were evaluated. The results showed that DEXA reduced the percentage of morphologically normal follicles, while CIMI prevented the deleterious effects caused by DEXA. In addition, DEXA negatively affected the stromal cellular density, while CIMI prevented these adverse effects. Ovaries cultured with DEXA and CIMI showed similar levels of mRNA for Bax, Bcl-2, and Caspase-3 compared to those cultured in control medium, while ovaries cultured with DEXA had increased expression of the above genes. Additionally, the ultrastructure of the ovaries cultured with CIMI was well preserved. Thus, the extract of CIMI was able to prevent the deleterious effects caused by DEXA on cultured mouse ovaries.
Subject(s)
Cimicifuga , Female , Animals , Mice , Caspase 3 , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/pharmacology , Cimicifuga/genetics , Cimicifuga/metabolism , Ovarian Follicle , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , RNA, Messenger/metabolism , Dexamethasone/toxicityABSTRACT
OBJECTIVE: To analyze the value of serum miRNA-122 expression in the diagnosis, severity, and prognosis of Acute Cerebral Infarction (ACI) and the correlation mechanism of serum miRNA-122 on the proliferation and apoptosis of vascular endothelial cells in ACI. METHOD: A total of 60 patients with ACI who were admitted to the emergency department of the Taizhou People's Hospital from January 1, 2019, to December 30, 2019, and 30 healthy controls during the same period were selected. General clinical data of all patients at admission were collected. Including age, sex, medical history, and inflammatory factors (C-Reactive Protein [CRP], Interleukin-6 [IL-6], Procalcitonin [PCT], Neutrophil Gelatinase-Associated Lipid carrier protein [NGAL]). The National Institutes of Health Stroke Scale (NIHSS) score at admission and short-term prognosis (the Modified Rankin Score [mRS]) score at 3 months after onset were recorded. The expression level of miRNA-122 in the serum of patients with ACI and normal controls was detected by reverse-transcription quantitative Real-Time Polymerase Chain Reaction (RT-QPCR), and the correlation between the expression level of miRNA-122 in the serum of patients with ACI and the level of inflammatory factors, NIHSS and mRS scores were analyzed. The expression levels of miRNA-122 in the serum of patients with ACI, normal people, and Human Umbilical cord Endothelial Cells (HUVECs) cultured in a blank control group were detected by RT-QPCR and statistically analyzed. MTT and flow cytometry was used to compare the proliferation and apoptosis of vascular endothelial cells in the miRNA-122 mimics and inhibitors transfection groups and the corresponding negative control group. The mRNA and protein levels of apoptosis-related factors Bax, Bcl-2, Caspase-3, and angiogenesis-related proteins Hes1, Notch1, Vascular Endothelial Growth Factors (VEGF), and CCNG1 were detected by RT-QPCR and Western blot. Bioinformatics methods predicted CCNG1 to be the target of miRNA-122, and the direct targeting relationship between CCNG1 and miRNA-122 was verified by a dual-luciferase reporting assay. RESULT: Serum miRNA-122 expression in patients with ACI was significantly higher than that in healthy controls, with an area under the receiver operating characteristic curve of 0.929, 95% Confidence Interval of 0.875â0.983, and an optimal cut-off value of 1.397. The expression levels of CRP, IL-6, and NGAL in patients with ACI were higher than those in healthy control groups, p < 0.05; miRNA-122 was positively correlated with CPR, IL-6, NIHSS score, and mRS score. At 48h and 72h, the proliferation rate of HUVECs cells in the miRNA-122 mimics group decreased and the apoptosis rate increased. Cell proliferation rate increased, and apoptosis rate decreased significantly in the groups transfected with miRNA-122 inhibitors. The mRNA and protein levels of pro-apoptotic factors Bax and caspase-3 were significantly increased in the miRNA-122 mimics transfection group, while those of anti-apoptotic factor Bcl-2 were significantly decreased compared to those of the control group. The expression of Bax and Caspase-3 decreased, and the expression of anti-apoptotic factor Bcl-2 increased in the transfected miRNA-122 inhibitors group. mRNA expression levels of Hes1, Notch1, VEGF, and CCNG1 in the miRNA-122 mimic transfected group were significantly decreased, while mRNA expression levels in the miRNA-122 inhibitors transfected group were significantly increased. Bioinformatics showed that there was a miRNA-122 binding site in the 3'UTR region of CCNG1, and dual luciferase assay confirmed that CCNG1 was the target of miRNA-122. CONCLUSION: Serum miRNA-122 increased significantly after ACI, which may be a diagnostic marker of ACI. miRNA-122 may be involved in the pathological process of ACI and is related to the degree of neurological impairment and short-term prognosis in patients with ACI. miRNA-122 may play a regulatory role in ACI by inhibiting cell proliferation, increasing apoptosis, and inhibiting vascular endothelial cell regeneration through the CCNG1 channel.
Subject(s)
Brain Ischemia , MicroRNAs , Stroke , Humans , MicroRNAs/genetics , Caspase 3/metabolism , Interleukin-6 , Vascular Endothelial Growth Factor A , Lipocalin-2 , Endothelial Cells/metabolism , bcl-2-Associated X Protein/metabolism , Cerebral Infarction , Apoptosis , C-Reactive Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Proliferation , RNA, MessengerABSTRACT
Due to their key role in the pathogenesis of cancer through the regulation of apoptosis, the B-cell leukemia/lymphoma-2 (BCL-2) family proteins have been an attractive target for cancer therapy for the past decades. Throughout the years, many Bcl-2 family inhibitors have been developed, with Venetoclax being now successfully used in treating hematological malignancies. Although their effectiveness in the treatment of solid tumors is yet to be established, some preclinical evidence indicates their possible clinical application. This review aims to summarize current data from completed clinical trials that used Bcl-2 protein family inhibitors as monotherapy or in combination with other agents for the treatment of solid malignancies. We managed to include clinical trials of various phases which analyze the pharmacokinetics and pharmacodynamics of the drugs, as well as the effectiveness and adverse effects. Active and recruiting clinical trials are also briefly presented and future prospects and challenges are discussed.
Subject(s)
Antineoplastic Agents , Hematologic Neoplasms , Leukemia, Lymphocytic, Chronic, B-Cell , Humans , Proto-Oncogene Proteins c-bcl-2/metabolism , Antineoplastic Agents/adverse effects , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Apoptosis , Bridged Bicyclo Compounds, Heterocyclic/therapeutic use , Bridged Bicyclo Compounds, Heterocyclic/pharmacologyABSTRACT
There is evidence of the existence of an intraovarian gonadotropin-releasing hormone (GnRH) system. There are also reports about the influence of extrinsic ovarian innervation in gonadal function. Therefore, it is interesting to study the relationship between ovarian sympathetic innervation and GnRH to shed light on possible physiological and pathophysiological implications. This work aimed to investigate whether noradrenergic stimulation of the superior mesenteric ganglion (SMG) can modify the levels of ovarian GnRH and cause functional and morphological changes in the gonad through the ovarian plexus nerve (OPN), during estrus and diestrus II in rats. The SMG-OPN-Ovary system and an ovary without extrinsic innervation were removed from Holtzman rats in estrus and diestrus II stages and placed in specially designed cuvettes containing Krebs-Ringer buffer. In the experimental groups, SMGs and denervated ovaries were stimulated with 10-6 M noradrenaline (NA). GnRH and progesterone levels (in the ovarian incubation medium) and the mRNA expression of 3beta-hydroxysteroid dehydrogenase (Hsd3b3), 20alpha-hydroxysteroid dehydrogenase (Akr1c18), Bax, and Bcl2 were analyzed. Histological studies of the ovaries were performed. In estrus, NA decreased GnRH levels in both experimental schemes. Furthermore, progesterone levels increased while the Akr1c18 expression and Bax/Bcl2 ratio decreased, without causing changes in ovarian morphology. In diestrus, the noradrenergic stimulation of the ganglion increased GnRH levels, decreased progesterone levels, and increased Akr1c18 expression and Bax/Bcl2 ratio. Follicles with histoarchitecture alterations and corpus luteum with signs of cell death were observed. In denervated ovaries, NA increased the levels of GnRH and progesterone. Furthermore, NA decreased the Bax/Bcl2 ratio and histological studies revealed signs compatible with a possible atretogenic effect. In conclusion, noradrenergic stimulation of the SMG-OPN pathway regulates ovarian cyclicity. The SMG modulates the cross-talk between NA and ovarian GnRH, protecting the ovary from atretogenic effects and luteal apoptosis during estrus while inducing luteal regression in the diestrus II.
Subject(s)
Ovary , Progesterone , Female , Rats , Animals , Ovary/metabolism , Progesterone/metabolism , Norepinephrine/metabolism , Norepinephrine/pharmacology , Gonadotropin-Releasing Hormone/metabolism , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/pharmacology , Rats, Sprague-Dawley , Proto-Oncogene Proteins c-bcl-2/metabolism , Hydroxysteroid Dehydrogenases/metabolismABSTRACT
The pathophysiology of sepsis may involve the activation of the NOD-type receptor containing the pyrin-3 domain (NLPR-3), mitochondrial and oxidative damages. One of the primary essential oxidation products is 8-oxoguanine (8-oxoG), and its accumulation in mitochondrial DNA (mtDNA) induces cell dysfunction and death, leading to the hypothesis that mtDNA integrity is crucial for maintaining neuronal function during sepsis. In sepsis, the modulation of NLRP-3 activation is critical, and mefenamic acid (MFA) is a potent drug that can reduce inflammasome activity, attenuating the acute cerebral inflammatory process. Thus, this study aimed to evaluate the administration of MFA and its implications for the reduction of inflammatory parameters and mitochondrial damage in animals submitted to polymicrobial sepsis. To test our hypothesis, adult male Wistar rats were submitted to the cecal ligation and perforation (CLP) model for sepsis induction and after receiving an injection of MFA (doses of 10, 30, and 50 mg/kg) or sterile saline (1 mL/kg). At 24 h after sepsis induction, the frontal cortex and hippocampus were dissected to analyze the levels of TNF-α, IL-1ß, and IL-18; oxidative damage (thiobarbituric acid reactive substances (TBARS), carbonyl, and DCF-DA (oxidative parameters); protein expression (mitochondrial transcription factor A (TFAM), NLRP-3, 8-oxoG; Bax, Bcl-2 and (ionized calcium-binding adaptor molecule 1 (IBA-1)); and the activity of mitochondrial respiratory chain complexes. It was observed that the septic group in both structures studied showed an increase in proinflammatory cytokines mediated by increased activity in NLRP-3, with more significant oxidative damage and higher production of reactive oxygen species (ROS) by mitochondria. Damage to mtDNA it was also observed with an increase in 8-oxoG levels and lower levels of TFAM and NGF-1. In addition, this group had an increase in pro-apoptotic proteins and IBA-1 positive cells. However, MFA at doses of 30 and 50 mg/kg decreased inflammasome activity, reduced levels of cytokines and oxidative damage, increased bioenergetic efficacy and reduced production of ROS and 8-oxoG, and increased levels of TFAM, NGF-1, Bcl-2, reducing microglial activation. As a result, it is suggested that MFA induces protection in the central nervous system early after the onset of sepsis.
Subject(s)
Mefenamic Acid , Sepsis , Animals , Rats , Male , Reactive Oxygen Species/metabolism , Mefenamic Acid/metabolism , Mefenamic Acid/pharmacology , Rats, Wistar , Inflammasomes/metabolism , Nerve Growth Factor/metabolism , Mitochondria , Sepsis/complications , Sepsis/drug therapy , DNA, Mitochondrial , Cytokines/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolismABSTRACT
The recurrence of Glioblastoma is partly attributed to the highly resistant subpopulation of glioma stem cells. A novel therapeutic approach focuses on restoring apoptotic programs in these cancer stem cells, as they are often deregulated. BH3-mimetics, targeting anti-apoptotic Bcl-2 family members, are emerging as promising compounds to sensitize cancer cells to antineoplastic treatments. Herein, we determined that the most abundantly expressed anti-apoptotic Bcl-2 family members, Bcl-xL and Mcl-1, are the most relevant in regulating patient-derived glioma stem cell survival. We exposed these cells to routinely used chemotherapeutic drugs and BH3-mimetics (ABT-263, WEHI-539, and S63845). We observed that the combination of BH3-mimetics targeting Bcl-xL with chemotherapeutic agents caused a marked increase in cell death and that this sensitivity to Bcl-xL inhibition correlated with Noxa expression levels. Interestingly, whereas co-targeting Bcl-xL and Mcl-1 led to massive cell death in all tested cell lines, down-regulation of Noxa promoted cell survival only in cell lines expressing higher levels of this BH3-only. Therefore, in glioma stem cells, the efficacy of Bcl-xL inhibition is closely associated with Mcl-1 activity and Noxa expression. Hence, a potentially effective strategy would consist of combining Bcl-xL inhibitors with chemotherapeutic agents capable of inducing Noxa, taking advantage of this pro-apoptotic factor.
Subject(s)
Antineoplastic Agents , Glioma , Humans , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Cell Line, Tumor , Apoptosis Regulatory Proteins/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis , Glioma/drug therapy , Neoplastic Stem Cells/metabolism , bcl-X Protein/metabolismABSTRACT
BACKGROUND: MicroRNAs (miRNAs) are involved in the progression of diverse human cancers. This work aimed to delve into how microRNA-135a-5p (miR-135a-5p) affects the biological behaviors of Breast Cancer (BC) cells. METHODS: Gene Expression Omnibus (GEO) datasets were used to analyze the expression differences of miR-135a-5p in cancer tissues of BC patients. Quantitative real-time PCR and western blot were conducted to detect miR-135a-5p and Bcl-2 Associated Athanogene (BAG3) expression levels in BC tissues and cells, respectively. The proliferation, migration, invasion, and cell cycle of BC cells were detected by cell counting kit-8 assay, BrdU assay, wound healing assay, transwell assay, and flow cytometry. The targeted relationship between miR-135a-5p and BAG3 mRNA 3'UTR predicted by bioinformatics was further testified by a dual-luciferase reporter gene assay. Pearson's correlation analysis was adopted to analyze the correlation between miR-135a-5p expression and BAG3 expression. The downstream pathways of BAG3 were analyzed by the LinkedOmics database. RESULTS: MiR-135a-5p was significantly down-regulated and BAG3 expression was significantly raised in BC tissues. MiR-135a-5p overexpression repressed the viability, migration and invasion of BC cells, and blocked cell cycle progression in G0/G1 phase while inhibiting miR-135a-5p worked oppositely. BAG3 was verified as a target of miR-135a-5p. Overexpression of BAG3 reversed the impacts of miR-135a-5p on the malignant biological behaviors of BC cells. The high expression of BAG3 was associated with the activation of the cell cycle, mTOR and TGF-ß signaling pathways. CONCLUSION: MiR-135a-5p regulates BAG3 to repress the growth, migration, invasion, and cell cycle progression of BC cells.
Subject(s)
Breast Neoplasms , MicroRNAs , 3' Untranslated Regions , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Breast Neoplasms/genetics , Bromodeoxyuridine , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Female , Gene Expression Regulation, Neoplastic/genetics , Humans , MicroRNAs/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
OBJECTIVES: This study aims to explore the effect of silencing Beclin-1 gene on autophagy and apoptosis of Benign Prostatic Hyperplasia (BPH) (BPH-1) cells under the condition of Androgen Deprivation (AD) and Autophagy Inhibition (AI). METHODS: Control group (BPH-1 group), empty carrier group (sh-RNA-BPH-1 group) and Beclin-1 silenced group (sh-Beclin1-BPH-1 group) were set. The Beclin-1 gene silencing efficiency was detected by RT-PCR and Western blot. Autophagic flux was monitored by GFP-LC3 cleavage assay and cell apoptosis was analyzed by flow cytometry. The protein expression levels of LC3, Caspase-3, PARP-1, Bcl-2, and Bax were detected by Western blot. RESULTS: The transfection of sh-Beclin-1 obviously down-regulated the expression of Beclin-1 at both mRNA and protein levels. Under the conditions of AD and AI, silencing of Beclin-1 restrained the autophagy of BPH-1 cells, as evidenced by a decreased number of autophagosomes and down-regulation of LC3-II protein (p < 0.001). The results of flow cytometry showed that the apoptotic rate of sh-Beclin-1 group was elevated significantly compared to the other two groups (p < 0.01). Western blot results showed that silencing of Beclin-1 promoted 89 kd fragmentation of PARP-1 (p < 0.001) and Caspase-3 activation (p < 0.01). Moreover, silencing of Beclin-1 resulted in declined Bcl-2 and augmented Bax protein expression in BPH-1 cells (p < 0.01), which ultimately led to a decreased Bcl-2/Bax ratio. CONCLUSIONS: The results indicated that the silencing of Beclin-1 gene hampered autophagy while activating apoptosis in BPH-1 cells. Thus, Beclin-1 may participate in an antagonistic relationship between autophagy and apoptosis in BPH.
Subject(s)
Prostatic Hyperplasia , Prostatic Neoplasms , Androgen Antagonists , Apoptosis/genetics , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Apoptosis Regulatory Proteins/pharmacology , Autophagy , Beclin-1/genetics , Beclin-1/metabolism , Beclin-1/pharmacology , Caspase 3/genetics , Caspase 3/metabolism , Caspase 3/pharmacology , Epithelial Cells/metabolism , Gene Silencing , Humans , Male , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Prostatic Hyperplasia/genetics , Prostatic Neoplasms/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , bcl-2-Associated X Protein/pharmacologyABSTRACT
OBJECTIVE: The purpose of this study was to investigate the cellular and molecular levels of apoptosis induction in three groups of male partners of infertile couples, one featuring subjects with normal sperm parameters and unexplained male infertility (UMI), one including men with abnormal sperm parameters, and one with fertile men as controls. METHODS: Twenty-five infertile men with abnormal sperm parameters and 25 men with UMI and normal sperm parameters were recruited as experimental group I and experimental group II; 25 fertile men were included as controls. The mRNA levels of Fas, Fas ligand, Caspase 8, Bax, and Bcl2 were measured in the three groups. The cellular rates of early and late apoptosis were assessed using annexin V and propidium iodide staining. RESULTS: The expression of Bax, Bcl2, and the Bax/Bcl2 ratio in experimental group I was significantly higher than that in experimental group II and controls. However, the Bax/Bcl2 ratio was less than 1 among all groups. No significant difference was found among study groups regarding the gene expression of Fas, Fas ligand, and Caspase 8. No significant difference was seen in early apoptotic rates of sperm among study groups. The highest number of necrotic sperm cells was detected in experimental group I. CONCLUSIONS: The findings showed that the external pathways of apoptosis were not activated in the absence of external stimuli of sperm apoptosis in ejaculated sperm. Regardless of fertility status, apoptosis gene induction in the internal pathway was associated with abnormalities in sperm motility and/or morphology in men with abnormal parameters.
Subject(s)
Infertility, Male , Sperm Motility , Male , Humans , Fas Ligand Protein/genetics , Fas Ligand Protein/metabolism , Caspase 8/genetics , Caspase 8/metabolism , Semen/metabolism , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolism , Infertility, Male/genetics , Spermatozoa , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Gene ExpressionABSTRACT
BACKGROUND: To our knowledge, there is no useful and accurate prognostic biomarker or biomarkers for patients with oral squamous cell carcinoma (OSCC), a tumor with uncertain biological behavior, and unpredictable clinical progress. The purposes of this study were: a) to determine the expresión profile of Connexin 43, Bcl-2, Bax, E-cadherin, and Ki67 in patients with OSCC; b) identify the GJCA1 rs12197797 genotypic composition. MATERIAL AND METHODS: A cross-sectional study using genomic DNA and biopsy samples extracted from the oral mucosa with/without OSCC, older than 18 years, both genders, attended at Facultad de Odontología, Universidad Nacional Córdoba. Immunostaining for Cx43, Bcl-2, Bax, E-cadherin, and Ki67 and genotyping GJA1 rs12197797 by RFLP were performed. Odds Ratio (95% CI), Spearman Coefficient were estimated. Mann-Whitney test was applied to analyze immunostaining between controls/cases (p <0.05 was set for statistical significance). RESULTS: GG (mutant) was the most frequent genotype in patients with OSCC diagnosis (53.2%) in relation to CC "healthy" genotype (p=0.00487; OR=7.33; CI95% [1.1-54.7]). And, the allele G (mutant) had a presence in 75.5% of OSCC patients. However, no significant association was observed between alleles C/G and diagnosis (p=0.0565). The heterozygous genotype was the most frequent in the patients of both groups Cx43 and E-cadherin markers were lower in OSCCs in relation to controls. Ki67 and Bcl-2 immunolabeling were high on OSCC, and Bax immunomarker was diminished in OSCC. CONCLUSIONS: We hypothesized that the oral epithelium losses Connexin 43 and E-cadherin in the membrane, which modifies cell differentiation. The Ki67 and Bcl2 overexpression would increase the cell density in the tissue, by promoting proliferation and decreasing apoptosis. And, this study shows evidence that patients who carry on allele G of GJA1rs12197797 could be at risk of developing OSCC.
Subject(s)
Carcinoma, Squamous Cell , Head and Neck Neoplasms , Mouth Neoplasms , Biomarkers, Tumor/genetics , Cadherins/genetics , Carcinoma, Squamous Cell/pathology , Connexin 43/genetics , Cross-Sectional Studies , Female , Humans , Ki-67 Antigen , Male , Mouth Neoplasms/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Squamous Cell Carcinoma of Head and Neck , bcl-2-Associated X ProteinABSTRACT
PURPOSE: The present study explored the role of melatonin in cisplatin-induced cardiac injury along with the possible role of brain-derived neurotrophic factor (BDNF) in melatonin-mediated effects. METHODS: Wistar rats were administered cisplatin (10 mg/kg), and cardiac injury was assessed by measuring the levels of cardiac troponin (cTnT) and lactate dehydrogenase (LDH-1).The extent of apoptosis was measured by measuring caspase-3 (pro-apoptotic) and Bcl-2 (anti-apoptotic) in hearts. The levels of BDNF, tumour necrosis factor α (TNF-α) and reduced glutathione were measured in heart. Melatonin (5 and 10 mg/kg) was administered for 15 days, and the role of BDNF was identified by co-administering BDNF inhibitor, ANA-12 (0.25 and 0.5 mg/kg). RESULTS: Melatonin attenuated cTnT and LDH-1 levels along with reduction in caspase-3 and increase in Bcl-2. It also increased cisplatin-induced decrease in BDNF, increase in TNF-α and decrease in reduced glutathione levels. Moreover, ANA-12 abolished the cardioprotective effects, anti-inflammatory and antioxidant effects of melatonin suggesting the role of BDNF in melatonin-mediated effects in cisplatin-induced cardiac injury. CONCLUSIONS: Melatonin is useful in cisplatin-induced cardiac injury, which may be due to an increase in BDNF, decrease in inflammation and increase in antioxidant activities.
Subject(s)
Melatonin , Animals , Antioxidants/metabolism , Antioxidants/pharmacology , Apoptosis , Brain-Derived Neurotrophic Factor/metabolism , Brain-Derived Neurotrophic Factor/pharmacology , Caspase 3/metabolism , Cisplatin/toxicity , Glutathione/metabolism , Melatonin/pharmacology , Melatonin/therapeutic use , Oxidative Stress , Proto-Oncogene Proteins c-bcl-2/metabolism , Rats , Rats, Wistar , Signal Transduction , Tumor Necrosis Factor-alpha/metabolismABSTRACT
The intracellular parasite Leishmania mexicana inhibits camptothecin (CPT)-induced apoptosis of monocyte-derived dendritic cells (moDC) through the down-regulation of p38 and JNK phosphorylation, while the kinase Akt is maintained active for 24 h. In addition, the infection of moDC with L. mexicana promastigotes increases the protein presence of the antiapoptotic protein Bcl-xL. In the present work, we aimed to investigate the role of Akt in the inhibition of apoptosis of moDC by L. mexicana and in the modulation of the expression of the antiapoptotic proteins Bcl-2, Mcl-1 and Bcl-xL. moDC were infected with L. mexicana metacyclic promastigotes and treated with CPT, an Akt inhibitor, or both and the mitochondrial outer membrane permeabilization (MOMP) and protein presence of active caspase 3, Bcl-2, Mcl-1 and Bcl-xL were evaluated. Our results show that the specific inhibition of Akt reverts the apoptosis protective effect exerted by L. mexicana on moDC reflected by a reduction in MOMP, caspase 3 activation, and upregulation of Bcl-xL. Interestingly, we also found that the infection of moDC with L. mexicana promastigotes induces a decrease in Bcl-2 along with an isoform change of Mcl-1, this independently to Akt activity. We demonstrated that Akt is deeply involved in the inhibition of apoptosis of moDC by L. mexicana.
Subject(s)
Leishmania mexicana , Apoptosis , Apoptosis Regulatory Proteins , Camptothecin/pharmacology , Caspase 3 , Dendritic Cells/parasitology , Leishmania mexicana/physiology , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-akt/metabolism , Proto-Oncogene Proteins c-akt/pharmacology , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-bcl-2/pharmacology , bcl-X Protein/metabolismABSTRACT
BACKGROUND: ROR2 is a tyrosine-kinase receptor whose expression is dysregulated in many human diseases. In cancer, ROR2 stimulates proliferation, survival, migration, and metastasis, and is associated with more aggressive tumor stages. The purpose of this work is to study the role of ROR2 in the chemoresistance of melanoma. METHODS: Gain- and loss-of-function experiments were used to study the biological function of ROR2 in melanoma. Cell death induced by chemotherapeutic drugs and BH-3 mimetics was evaluated using crystal violet cytotoxicity assays and annexin V/propidium iodide staining. Western blots were used to evaluate the expression of proteins implicated in cell death. The differences observed between cells with manipulation of ROR2 levels and control cells were evaluated using both Student's t-test and ANOVA. RESULTS: We describe that ROR2 contributes to tumor progression by enhancing the resistance of melanoma cells to both chemotherapeutic drugs and BH-3 mimetics. We demonstrate that ROR2 reduced cell death upon treatment with cisplatin, dacarbazine, lomustine, camptothecin, paclitaxel, ABT-737, TW-37, and venetoclax. This effect was mediated by the inhibition of apoptosis. In addition, we investigated the molecular mechanisms implicated in this role of ROR2. We identified the MDM2/p53 pathway as a novel target of ROR2 since ROR2 positively regulates MDM2 levels, thus leading to p53 downregulation. We also showed that ROR2 also upregulates Mcl-1 and Bcl2-xL while it negatively regulates Bax and Bid expression. The effect of ROR2 on the expression of these proteins is mediated by the hyperactivation of ERK. CONCLUSIONS: These results demonstrate that ROR2 contributes to melanoma progression by inhibiting apoptosis and increasing chemoresistance. These results not only position ROR2 as a marker of chemoresistance but also support its use as a novel therapeutic target in cancer.