Apoptotic Gene Expression Research Articles

Anti-proliferative Effects of Ocimum basilicum Leaf Aqueous Extract on Colon Cancer Cell Lines and the Expression of Apoptotic Genes

Background: Colon cancer is one of the most diagnosed cancers in the modern world. Chemotherapy is one of the most relevant treatments for colon cancer, but this method of treatment has severe and sometimes irreversible side effects. Therefore, alternative treatments with fewer side effects are highly considered. Natural and herbal medicine has been proven to be a great source for these alternative treatment methods. Objectives: This study aimed to investigate the anti-proliferative and cytotoxic effects of Ocimum basillicum (OB) leaf aqueous extract on human colon cancer cell lines LS174T and COLO205 and the expression of apoptotic genes. Methods: Methyl thiazol tetrazolium (MTT) assay was performed to assess the cytotoxic effects of our extract on the aforementioned cell lines. The changes in the expression of BAX and BAD were analyzed by qPCR following the treatment of cells with our extract. Results: The results of the MTT assay indicated that the extract had a cytotoxic effect and could reduce the population of live cancer cells, especially LS174T cells. The expression of BAX and BAD genes showed a significant increase in qPCR analysis in both cell lines, especially in the LS174T cells. Conclusions: Our results showed that the extract had cytotoxic, anti-proliferative, and pro-apoptotic characteristics on previously mentioned cell lines, especially on the LS 174T cells.

Cooperative Treatment of Gastric Cancer Using B7-H7 siRNA and Docetaxel; How Could They Modify Their Effectiveness?

Despite the high prevalence of gastric cancer (GC), drug resistance is a major problem for effective chemotherapy. B7-H7 is a novel member of the B7 superfamily and is expressed in most common cancers. However, the role of B7-H7 on the aggressiveness of GC and chemosensitivity has remained unknown. Therefore, this study was designed to assess the effect of B7-H7 suppression using small interference RNA (siRNA) in combination with docetaxel on GC cells. MTT test was applied to determine the IC50 of docetaxel and the combined effect of B7-H7 siRNA and docetaxel on the viability of the MKN-45 cells. To determine B7-H7, BCL-2, BAX, and caspase-3-8-9 genes expression, qRT-PCR was performed. Furthermore, flow cytometry was applied to evaluate apoptosis and the cell cycle status. Finally, to evaluate the effect of this combination therapy on migratory capacity and colony-forming ability, wound healing assay and colony formation test were employed, respectively. B7-H7 suppression increased the chemo-sensitivity of MKN-45 cells to docetaxel. The expression of B7-H7 mRNA was reduced after using B7-H7 siRNA and docetaxel in MKN-45 GC cells. Also, B7-H7 suppression alongside docetaxel reduced cell migration and colony formation rate, arrested the cell cycle at the G2-M phase, and induced apoptosis by modulating the expression of apoptotic target genes. B7-H7 plays a significant role in the chemo-sensitivity and pathogenesis of GC. Therefore, B7-H7 suppression, in combination with docetaxel, may be a promising therapeutic approach in treating GC.

Cannabis smoke condensate induces human gingival epithelial cell damage through apoptosis, autophagy, and oxidative stress

ObjectivesThis study aims to investigate the effects of cannabis smoke condensate (CSC) on the adhesion, growth, and signaling pathways of human gingival epithelial cells. DesignThe effects of CSC on cell shape and adhesion, and viability were evaluated after 30 min, 60 min, 2 h, and 24 h of exposure using microscopic observation, cell metabolic activity, and lactate dehydrogenase activity assays. The effects of CSC on cell apoptosis, necrosis, autophagy, and oxidative stress were determined through flow cytometry, while apoptotic and autophagic gene expression were identified via an RT2-PCR array. Phosphorylated signaling pathway proteins were measured using flow cytometry. ResultsCSC deregulated gingival epithelial cell shape and adhesion, decreased cell viability, and increased lactate dehydrogenase release. Its toxic effects included apoptosis, autophagy, and oxidative stress. Moreover, it modulated seven specific apoptotic and six autophagic genes. Furthermore, it decreased phosphorylation in signaling proteins, such as STAT5, ERK12, P38, and nuclear factor κB. ConclusionsCSC has notable adverse effects on gingival epithelial cells. This finding indicates that cannabis smoke could impair gingival epithelial cell innate immune function, leading to gingivitis and periodontitis. Oral health professionals may need to document observed modifications in the oral cavity of patients who smoke cannabis and consider these potential changes during clinical care.

Effect of Fluorouracil Combined With Harmine on Induction of Apoptosis in Apoptosis Induction in Pancreatic Cancer Cells (AsPC-1) and the Expression of Apoptotic Genes (BAX, P53, Caspase-3, and Caspase-9)

Objective: Pancreatic cancer is one of the deadliest cancers related to the digestive system in the world. Due to the increasing resistance of cancer cells to chemotherapy and its side effects, there is a need for drugs with fewer side effects and natural origin. In this study, the effect of harmine combined with fluorouracil on apoptosis induction in pancreatic cancer cells (AsPC-1) and the expression of apoptotic genes was investigated. Methods: In this experimental study, MTT assay was used to study the cytotoxic effects. Annexin V/propidium iodide test and DAPI staining method were used to determine the type of cell death. The expression of apoptotic genes (P53, Bax, Caspase-3, and Caspase-9) was measured by real-time PCR. The data were analyzed using one-way ANOVA in SPSS software, version 16. The significance level was set at 0.05. Results: The results of MTT assay showed that the use of harmine at 20 μg/mL concentration combined with 10 μg/mL fluorouracil significantly reduced the growth (IC50) of AsPC-1 cells. The results of DAPI staining and annexin V/propidium iodide test showed the induction of apoptosis in treated samples. In addition, the results of real-time PCR showed that the expression of BAX, P53, Caspase-3 and Caspase-9 genes increased in the treated groups compared to the control group. Conclusion: The combined use of harmine and fluorouracil can induce apoptosis in pancreatic cancer cells due to high cytotoxicity and reduce the concentration of chemotherapy drugs. Therefore, this method can be used along with other treatment methods for pancreatic cancer.

O-265 Luteinizing hormone is able to protect reproductive health in cancer patients

Abstract Study question Is luteinizing hormone (LH) able to protect ovarian follicles against cyclophosphamide (CPM)-induced damage in women? Summary answer LH significantly reduces primordial follicle (PMF) loss in ovarian cortical strips cultured in vitro with phosphoramide mustard (PM), the active metabolite of CPM. What is known already Cancer therapies are cause of premature ovarian insufficiency (POI) in female patients, since they induce a severe reduction of PMFs. For this reason, several compounds have been analyzed as adjuvant therapies to protect the ovarian reserve without interfering with cancer treatment on tumor cells. We recently demonstrated the protective effect of LH against ovotoxicity induced by PM and cisplatin on the ovary of prepuberal and adult mice. These results suggest the possibility to use LH prior or in concomitance with anticancer drugs in order to preserve oocytes in human patients, therefore preventing the early onset of menopause and/or infertility. Study design, size, duration Ovarian cortical tissues were collected from nine patients (age ± SD: 15.33 ± 4.50) who have cryopreserved their tissue before receiving anticancer treatment. For each patient, ovarian cortical strips were thawed and randomly assigned to the experimental conditions: Control (CTRL), PM and PM+LH. LH was added 1 hour before the treatment with PM. Samples were analyzed after 8, 16, 24 and 48 hrs of treatment. Participants/materials, setting, methods Ovarian cortial strips were placed onto culture plate insert and cultured for a maximum of 48 hours, in αMEM with 10% Serum Substitute Supplement with/out 200mIU/ml LH and/or 10μM PM. The samples were processed for: - Histology, for PMFs and primary follicles (PFs) analysis; - Immunohistochemistry, for the expression of protein involved in DNA damage, apoptosis and follicle activation; - Real-Time PCR, for the expression of apoptotic and inflammation related genes. Main results and the role of chance The follicle density in the untreated group varied from 233.03 to 3420.27 PMFs/mm3 between the nine patients analysed. Relative follicular density (%) was performed to analyse statistical differences between the groups. Relative PMFs density (%) was significantly reduced in PM vs CTRL either after 24 and 48 hrs, while this reduction was significantly counteracted by LH (24 hrs: CTRL=76.19±2.12; PM = 44.95±5.06; PM+LH=73.97±11.64. 48 hrs: CTRL=66.47±3.96; PM = 36.76±3.96; PM+LH=55.74±8.72). To investigate the mechanism underlying the observed effects, the expression of markers involved in DNA damage (gH2AX), apoptosis (Cleaved caspase 3, NOXA, PUMA), follicle activation (p-AKT, FOXO3a), cell cycle arrest (p21, Ki67), and inflammation (IL1β, TNFα) were analysed. The results showed that LH did not prevent DNA damage induced by PM, since gH2AX positive oocytes were seen both in PM and PH+LH group at 16h; at 24hrs however we observed a downregulation of the gH2AX expression in the PH+LH group (PM@ 100% vs PM+LH@35%). LH also counteracts the activation of chemotherapy-induced apoptotic processes, by reducing the levels of pro-apoptotic factors such as NOXA, PUMA and CC3, and follicles activation lowering AKT-FOXO3a signaling axis. Moreover, PM treatment creates a proinflammatory microenvironment, as shown by increased IL1β and TNFα gene expression, partially counteracted by LH pretreatment. Limitations, reasons for caution Experiments are carried out in vitro; the functionality of the cortex in xenografts should be evaluated. Careful evaluation of which patients might use this protector is needed. Wider implications of the findings These findings demonstrate that LH is able to reduce PMFs loss in human ovarian cortex exposed to PM. These results encourage thinking about the use of the hormone as a ferto-protector agent in clinical trials to prevent the premature onset of menopause and/or infertility in women undergoing anticancer treatments. Trial registration number not applicable

Relationship between embryo development and apoptotic gene expression of cumulus cells in poor responders and polycystic ovary syndrome

Research questionIs there a relationship between embryo quality, pregnancy rates and apoptotic gene expression in cumulus cells of oocytes collected from patients with poor ovarian response and polycystic ovary syndrome? DesignFifty infertile couples who underwent assisted reproductive technology treatment were included in the study (Approval date 4 February 2020, number 03). The patients were divided into four group: control (n = 9; 90 oocytes), unexplained infertility (n = 8; 86 oocytes), polycystic ovary syndrome (PCOS) (n = 6; 137 oocytes) and poor ovarian response (POR) (n = 27; 124 oocytes). Cumulus cells were isolated individually from 437 oocytes obtained. Intracytoplasmic sperm injection was undertaken on 365 mature oocytes. The embryos were monitored. Caspase-3, Bax and Bcl-2 gene expressions of the cumulus cells were measured by real-time polymerase chain reaction. ResultsA significant and negative correlation was found between Bax and Bcl-2 expressions of the cumulus cells of poor-quality embryos. The increase in Caspase-3 gene expression in the POR group statistically decreases the pregnancy rates. Fertilization and good-quality embryo development of 365 oocytes whose cumulus cells were examined, however, were not associated with apoptotic gene expression. The Bax/Bcl-2 ratio was found to be significantly lower in cumulus cells of mature oocytes. ConclusionsOur results demonstrated no significant associations between fertilization, quality embryo development and apoptotic gene expression. Bax expression and the Bax/Bcl-2 ratio are high in immature oocyte cumulus cells has shown us that the apoptotic process may begin when the cumulus–oocyte connection exists.

Molecular Characterization and Expression Analysis of Glutaredoxin 5 in Black Tiger Shrimp (Penaeus monodon) and Correlation Analysis Between the SNPs of PmGrx5 and Ammonia-N Stress Tolerance Trait

Glutaredoxins (Grxs) are glutathione-dependent oxidoreductases that belong to the thioredoxin (Trx) superfamily and are an essential part of the redox system in living organisms. However, there is a serious lack of sequence information and functional validation associated with Grxs in crustaceans. In this study, a new Grx gene (PmGrx5) was identified and characterized in black tiger shrimp (Penaeus monodon). The full-length cDNA of PmGrx5 is 787 bp and consists of 114 bp 5′-UTR, 232 bp 3′-UTR, and 441 bp ORF, encoding a hypothetical protein of 146 amino acids. The putative PmGrx5 protein is 16.27 kDa with a theoretical isoelectric point of 5.90. Sequence alignment showed that PmGrx5 had the highest amino acid sequence homology with Grx5 from Penaeus vannamei at 98.63% and clustered with Grx5 from other crustaceans. Quantitative real-time PCR (qRT-PCR) analysis showed that PmGrx5 was expressed in all tissues examined, with a higher expression in the testis, stomach, lymphoid organ, and gill. PmGrx5 was continuously expressed during development, with the highest expression in zoea I. Ammonia-N stress and bacterial infection both differentially upregulated PmGrx5 expression in the hepatopancreas and gill. In addition, when PmGrx5 was inhibited, the expression of some other antioxidant enzymes was upregulated at the beginning of ammonia-N stress, but as the stress time increased, the expression of antioxidant enzymes was inhibited, the expression of apoptotic genes was increased, and the GSH content was significantly reduced. Inhibition of PmGrx5 led to a greater risk of oxidative damage in shrimp. In addition, the relationship between SNPs in exons of the PmGrx5 gene and tolerance to ammonia-N stress was identified and analysed. A total of nine SNPs were successfully identified, eight of which were significantly associated with ammonia and nitrogen stress tolerance trait in shrimp (P < 0.05). The present study shows that PmGrx5 is involved in redox regulation and plays an important role in shrimp resistance to marine environmental stresses. Meanwhile, this study will provide a basis for molecular marker breeding in shrimp.

The Effect of Dual Bioactive Compounds Artemisinin and Metformin Co-loaded in PLGA-PEG Nano-particles on Breast Cancer Cell lines: Potential Apoptotic and Anti-proliferative Action.

The most prevalent malignancy among women is breast cancer. Phytochemicals and their derivatives are rapidly being recognized as possible cancer complementary therapies because they can modify signaling pathways that lead to cell cycle control or directly alter cell cycle regulatory molecules. The phytochemicals' poor bioavailability and short half-life make them unsuitable as anticancer drugs. Applying PLGA-PEG NPs improves their solubility and tolerance while also reducing drug adverse effects. According to the findings, combining anti-tumor phytochemicals can be more effective in regulating several signaling pathways linked to tumor cell development. The point of the study was to compare the anti-proliferative impacts of combined artemisinin and metformin on cell cycle arrest and expression of cyclin D1 and apoptotic genes (bcl-2, Bax, survivin, caspase-7, and caspase-3), and also hTERT genes in breast cancer cells. T-47D breast cancer cells were treated with different concentrations of metformin (MET) and artemisinin (ART) co-loaded in PLGA-PEG NPs and free form. The MTT test was applied to assess drug cytotoxicity in T47D cells. The cell cycle distribution was investigated using flow cytometry and the expression levels of cyclin D1, hTERT, Bax, bcl-2, caspase-3, and caspase-7, and survivin genes were then determined using real-time PCR. The findings of the MTT test and flow cytometry revealed that each state was cytotoxic to T47D cells in a time and dose-dependent pattern. Compared to various state of drugs (free and nano state, pure and combination state) Met-Art-PLGA/PEG NPs demonstrated the strongest anti-proliferative impact and considerably inhibited the development of T-47D cells; also, treatment with nano-formulated forms of Met-Art combination resulted in substantial downregulation of hTERT, Bcl-2, cyclin D1, survivin, and upregulation of caspase-3, caspase-7, and Bax, in the cells, as compared to the free forms, as indicated by real-time PCR findings. The findings suggested that combining an ART/MET-loaded PLGA-PEG NP-based therapy for breast cancer could significantly improve treatment effectiveness.

Silica Nanoparticle–Induced Reproductive Toxicity in Male Albino Rats via Testicular Apoptosis and Oxidative Stress

Amorphous silica nanoparticles (SiNPs) are being utilized in different fields such as medicine, cosmetics, and foods. However, the causes and mechanisms underlying SiNP testicular damage remain largely unclear. In the present study, we aimed to investigate this issue. Thirty male rats were randomly divided into three groups: control group (n = 10), 500 ppm SiNP–treated group (n = 10), and 1000 ppm SiNP–treated group (n = 10). SiNPs were given orally in drinking water for 30 days. Micronucleus assay was performed on blood RBCs. The concentrations of testicular malondialdehyde (MDA) and glutathione (GSH) and catalase (CAT) activity were measured. Moreover, the histopathological alterations and the expression of apoptotic (caspase-3) and pro-inflammatory and oxidative stress markers (iNOS) in testes and epididymis were analyzed and compared between the three groups. The results showed an increased level of micronucleus frequencies in the 1000 ppm–treated group, as well as increased levels of MDA and decreased activity of CAT and GSH content in testicular tissues in the 1000 ppm–treated group, suggesting DNA damage and oxidative stress mechanisms. Also, there were significant testicular histopathological alterations in this group. Furthermore, 1000-ppm SiNPs could enhance testicular apoptosis, inflammation, and oxidative stress by increasing the expression of apoptotic, pro-inflammatory, and oxidative stress genes including caspase 3 and iNOS in the examined tissue. The lower concentration of SiNPs did not produce any significant biochemical, histopathological, or immunohistochemical alterations whereas 1000-ppm SiNPs resulted in significant testicular changes by exacerbating apoptotic, inflammatory, and oxidative stress–mediated testicular damage.

Cedrus libani tar prompts reactive oxygen species toxicity and DNA damage in colon cancer cells.

Many chemotherapeutic drugs used in cancer treatment have anticancer properties by inducing reactive oxygen species (ROS). However, the same effect occurs in normal cells, limiting the availability of these drugs. Therefore, studies on the detection of new herbal anticancer agents that have selective effects on cancer cells are of great importance. The aim of this study is to investigate the metabolite profile of Cedrus libani tar and its mechanism of anticancer effect on colon cancer cells. Effect of cedar tar on cells (12 cancers and 5 normal cell lines) viability was determined by MTT, apoptosis induction was determined by Annexin-V, ROS and MMP determined by flow cytometry assay. Cleaved caspase-8, 9 and Ɣ-H2AX expression determined by western blot. Apoptotic and antioxidant genes expression level determined by qPCR. Metabolite profiling was performed with LC-MS/MS and GC-MS. Cedar tar showed the highest cytotoxic effect among cancer cells in colon cancer (HCT-116, IC50: 30.4μg/mL) and its toxic effect on normal cells (HUVEC, IC50: 74.07μg/mL) was less than cancer cell. Cedar tar increases ROS production in colon cancer cells. The metabolite profile of the cedar tar contains high amounts of metabolites such as fatty acids mainly (Duprezianene, Himachalene and Chamigrene), phenolic compounds (mostly Coumarin, p-coumaric acid, Vanillic acid and tr-Ferulic acid etc.) and organic acids (mainly 3-oh propanoic acid, 2-oh butyric acid and 3-oh isovaleric acid etc.). As a result, it has been found that cedar tar has the potential to be used in the treatment of colon cancer.

Effect of Leptin and IGF-I Supplementation on in vitro Maturation and Expression of Apoptotic Genes in Goat Oocytes

Background: The quality of gametes is a crucial factor for the successful development of an embryo. The potential of oocyte development is acquired during oogenesis by many changes at the molecular and cellular levels. The present study explores the effect of leptin and IGF-I supplementation on in vitro oocyte maturation and the expression of apoptotic genes in matured goat oocytes. Methods: In vitro maturation of abattoir-derived oocytes was in maturation media having supplements as the group I (control), group II (leptin 20 ng/ml) and group III (IGF-I 100 ng/ml). From these oocytes, RNA was isolated and expressions of apoptotic genes were studied. Result: There was no significant difference found between cumulus expansion and also in nuclear maturation in groups I, II, III. The relative expression of BAX gene was 0.65 fold lower in group II and 0.42 fold lower in group III as compared to group I whereas relative expression of Mcl-1 gene was 2.55 fold higher in group II and 0.31 fold lower in group III as compared to group I.

Hesperidin Attenuates Oxidative Stress, Inflammation, Apoptosis, and Cardiac Dysfunction in Sodium Fluoride-Induced Cardiotoxicity in Rats.

Excessive fluoride intake has been reported to cause toxicities to brain, thyroid, kidney, liver and testis tissues. Hesperidin (HSP) is an antioxidant that possesses anti-allergenic, anti-carcinogenic, anti-oxidant and anti-inflammatory activities. Presently, the studies focusing on the toxic effects of sodium fluoride (NaF) on heart tissue at biochemical and molecular level are limited. This study was designed to evaluate the ameliorative effects of HSP on toxicity of NaF on the heart of rats in vivo by observing the alterations in oxidative injury markers (MDA, SOD, CAT, GPX and GSH), pro-inflammatory markers (NF-κB, IL-1β, TNF-α), expressions of apoptotic genes (caspase-3, -6, -9, Bax, Bcl-2, p53, cytochrome c), levels of autophagic markers (Beclin 1, LC3A, LC3B), expression levels of PI3K/Akt/mTOR and cardiac markers. HSP treatment attenuated the NaF-induced heart tissue injury by increasing activities of SOD, CAT and GPx and levels of GSH, and suppressing lipid peroxidation. In addition, HSP reversed the changes in expression of apoptotic (caspase-3, -6, -9, Bax, Bcl-2, p53, cytochrome c), levels of autophagic and inflammatory parameters (Beclin 1, LC3A, LC3B, NF-κB, IL-1β, TNF-α), in the NaF-induced cardiotoxicity. HSP also modulated the gene expression levels of PI3K/Akt/mTOR signaling pathway and levels of cardiac markers (LDH, CK-MB). Overall, these findings reveal that HSP treatment can be used for the treatment of NaF-induced cardiotoxicity.

Isorhamnetin: a flavonoid, attenuated doxorubicin-induced testicular injury via regulation of steroidogenic enzymes and apoptotic signaling gene expression in male rats.

BackgroundMale reproductive damage is one of the most adverse side effects of doxorubicin (DOX). Isorhamnetin is a natural flavonoid, which displays remarkable antioxidant potential.ObjectiveThe current research was designed to assess the protective effects of Isorhamnetin against DOX-instigated testicular damages.MethodsAdult male Wistar rats (n=32) were divided into 4 groups: control, DOX (3 mg/kg i.p. 3 doses each after 1 week), DOX + Isorhamnetin (3 mg/kg 3 doses each after 1 week +10 mg/kg i.p. daily for 28 days, respectively), and Isorhamnetin (10 mg/kg i.p. per day). After 28 days of treatment, biochemical, spermatogenic, steroidogenic, hormonal, proapoptotic, antiapoptotic, and histopathological parameters were estimated.ResultsDOX exposure significantly decreased the activity of acid phosphatase, lactate dehydrogenase, and gamma-glutamyl transferase. Furthermore, DOX substantially decreased the activities of antioxidant enzymes, i.e. catalase, superoxide dismutase, glutathione reductase, and glutathione peroxidase along with protein content, whereas it increased the malondialdehyde level. It also reduced sperm progressive motility, viability, the number of hypoosmotic tail swelled spermatozoa, and epididymis sperm count and increased the sperm morphological anomalies (head, midpiece, and tail). Besides, it decreased the levels of follicle-stimulating hormone, luteinizing hormone, and plasma testosterone and lowered the expression of steroidogenic enzymes (3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase, and steroidogenic acute regulatory protein) and testicular antiapoptotic marker (B-cell lymphoma 2) but increased the expression of proapoptotic markers (BCL2-associated X protein and caspase-3) along with histopathological impairments. However, isorhamnetin prevented all the damages caused by DOX.ConclusionConclusively, Isorhamnetin can be used as a powerful mitigating agent to avert DOX-induced testicular damages.

Single intratesticular injection of blood-serum-derived exosomes can potentially alleviate testopathy following testicular torsion.

BackgroundTesticular torsion (TT) is an acute inflammatory process leading to male infertility. Today, anti‐inflammatory effects of exosomes derived from blood serum are used in various laboratory procedures. In the present study, the anti‐inflammatory effects of blood‐serum‐derived exosomes in treatment of acute inflammation following TT in mice were evaluated.Materials and MethodsEighteen male mice were grouped as healthy control, TT, and TT + exosome. TT was induced surgically, and exosomes were extracted from blood serum and administrated by a single intratesticular injection (10 IU). Malondialdehyde (MDA) and Griess assays were used to evaluate the level of oxidative stress. Sperm indices, testosterone (Tes), and apoptotic gene expression (p‐53, Bcl2, and Caspase‐3) were also assessed. H&E and immunohistochemistry (IHC) stainings were used for histopathological investigations. Data analysis was applied by SPSS (v.19) software.ResultsOxidative stress and apoptotic genes expression were increased significantly (p < 0.05) in TT group compared with control. Sperm parameters and Tes were significantly increased, and expression of apoptotic genes was significantly reduced in TT + exosome group (p < 0.05).ConclusionSince the blood‐serum‐derived exosomes have anti‐inflammatory features, the intratesticular application of blood‐serum‐derived exosomes can be used clinically in acute phase of orchitis following TT to inhibit testicular inflammation.

Thymoquinone Alterations of the Apoptotic Gene Expressions and Cell Cycle Arrest in Genetically Distinct Triple-Negative Breast Cancer Cells.

Breast cancer (BC) is the most common cancer in women worldwide, and it is one of the leading causes of cancer death in women. triple-negative breast Cancer (TNBC), a subtype of BC, is typically associated with the highest pathogenic grade and incidence in premenopausal and young African American (AA) women. Chemotherapy, the most common treatment for TNBC today, can lead to acquired resistance and ineffective treatment. Therefore, novel therapeutic approaches are needed to combat medication resistance and ineffectiveness in TNBC patients. Thymoquinone (TQ) is shown to have a cytotoxic effect on human cancer cells in vitro. However, TQ’s mode of action and precise mechanism in TNBC disease in vitro have not been adequately investigated. Therefore, TQ’s effects on the genetically different MDA-MB-468 and MDA-MB-231 human breast cancer cell lines were assessed. The data obtained show that TQ displayed cytotoxic effects on MDA-MB-468 and MDA-MB-231 cells in a time- and concentration-dependent manner after 24 h, with IC50 values of 25.37 µM and 27.39 µM, respectively. Moreover, MDA-MB-231 and MDA-MB-468 cells in a scratched wound-healing assay displayed poor wound closure, inhibiting invasion and migration via cell cycle blocking after 24 h. TQ arrested the cell cycle phase in MDA-MB-231 and MDA-MB-468 cells. The three cell cycle stages in MDA-MB-468 cells were significantly affected at 15 and 20 µM for G0/G1 and S phases, as well as all TQ concentrations for G2/M phases. In MDA-MB-468 cells, there was a significant decrease in G0/G1 phases with a substantial increase in the S phase and G2/M phases. In contrast, MDA-MB-231 showed a significant effect only during the two cell cycle stages (S and G2/M), at concentrations of 15 and 20 µM for S phases and all TQ values for G2/M phases. The TQ effect on the apoptotic gene profiles indicated that TQ upregulated 15 apoptotic genes in MDA-MB-231 TNBC cells, including caspases, GADD45A, TP53, DFFA, DIABLO, BNIP3, TRAF2/3, and TNFRSF10A. In MDA-MB-468 cells, 16 apoptotic genes were upregulated, including TNFRSF10A, TNF, TNFRSF11B, FADD TNFRSF10B, CASP2, and TRAF2, all of which are important for the apoptotic pathway andsuppress the expression of one anti-apoptotic gene, BIRC5, in MDA-MB-231 cells. Compared to MDA-MB-231 cells, elevated levels of TNF and their receptor proteins may contribute to their increased sensitivity to TQ-induced apoptosis. It was concluded from this study that TQ targets the MDA-MB-231 and MDA-MB-468 cells differently. Additionally, due to the aggressive nature of TNBC and the lack of specific therapies in chemoresistant TNBC, our findings related to the identified apoptotic gene profile may point to TQ as a potential agent for TNBC therapy.

Apoptotic qPCR gene expression array analysis demonstrates proof-of-concept for rapid blastocoel fluid-conditioned media molecular prediction.

Successful identification of transcriptomic biomarkers within human IVF embryos may enhance implantation prediction and provide insights not available through conventional embryo biopsy genomic analysis. We demonstrate proof-of-concept for a methodology to assess overall embryo gene expression using qPCR with blastocoel fluid-conditioned media by examining the comparative presence of apoptotic genes. Blastocoel fluid-conditioned media were collected from 19 embryos (11 euploid) following trophectoderm biopsy of day-5 ICSI-IVF blastocysts. Media were assessed for apoptotic gene expression via qPCR. Statistical analysis of gene expression was conducted via Wilcoxon Signed-Ranks test (overall expression), multivariate ANOVA (functional gene groups), and chi-square test of independence (gene level). A significantly higher overall apoptotic gene expression within euploid versus aneuploid embryos (p = 0.001) was observed. There was significantly (p = 0.045) higher expression of pro-apoptotic genes between implanted and not implanted embryos. Pro- vs. anti-apoptotic gene expression from all euploid embryos approached significance (p = 0.053). The ploidy status-based claim is further substantiated at the gene level with significantly higher expression of BBC3 (p = 0.012) and BCL2L13 (p = 0.003) in euploid embryos compared to aneuploid embryos. In this preliminary study, we demonstrate that (1) qualitative analysis of blastocoel fluid-conditioned media gene expression is possible, (2) global trends of expression are potentially related to clinical outcomes, and (3) gene-level expression trends exist and may be another viable metric for comparative expression between samples. The presence of statistical significance within analyses conducted with this sample size warrants a larger investigation of blastocoel fluid-conditioned media as an additional beneficial predictive tool for future IVF cases.

EAE suppression caused by Δ-8-THC treatment is due to downregulation of CNS infiltrating CD4+ T-cells, proinflammatory cytokines and apoptotic genes expression

Abstract Δ-8-THC a naturally occurring cannabinoid found in trace amounts in Cannabis plants. It possesses potent anti-inflammatory properties along with several other medical benefits. Δ-8-THC has been shown to induce immune system homeostasis particularly in individuals with autoimmune disorders by reducing the activity of T cells and B cells. In current study, we used the experimental model of MS to better understand how Δ-8-THC suppresses neuroinflammation in immune cell-mediated disease called experimental autoimmune encephalomyelitis (EAE). Δ-8-THC administration reduced significantly the clinical severity score with an increase in body weight by suppression of Th17 cells. In the CNS, there was decreased expression of pro-inflammatory cytokine IL-17, an increase in brain infiltrating CD4+ T cells and B cells, while anti-inflammatory phenotype such as FoxP3 was perturbed. Based on preliminary miRNA sequencing data and in silico examination of differential expression of miRNAs, we found that the decrease in neuroinflammation following treatment may be due to up-regulation of miRs, mmu-miR-129–3p, mmu-miR-129-5p, mmu-miR-125-5p and mmu-miR-193-3p as well as down-regulation of mmu-miR-21-3p, mmu-miR-142-5p and mmu-miR147-3p in immune cells in the CNS. Target genes were predicted by TagetScan, miRbase, miRDB. The target genes associated with upregulated miRs included Hrk, CHMP2b, Arid3a and Dcaf7 which are mostly involved in apoptosis, autophagy and migration, while genes associated with down-regulated miRNAs were Abi2, ZFPM2, NDUFA4 that are mostly involved in cell growth, proliferation and differentiation. In summary, Δ-8-THC treatment may offer novel strategies for treatment of neuroinflammation by targeting miRs. Supported by NIH P01AT003961, P20GM103641, R01AI129788, R01 ES030144, R01 AI160896 and R01AI123947

Delivery of miRNA-126 through folic acid-targeted biocompatible polymeric nanoparticles for effective lung cancer therapy

Objective: Nanoparticle-based drug delivery systems (DDSs) have been playing a considerable role in the eradication of cancer. In this experimental study, we designed and synthesized folic acid (FA)-decorated chitosan (CS) nanocarrier for targeted delivery of miR-126 (as a therapeutic agent) to lung cancer A549 cells. Materials and methods: Therefore, the FA-CS-miR-126 nano-complex was perfectly developed and characterized by various analytical devices such as Fourier transform infrared (FT-IR) and dynamic light scattering (DLS) spectroscopies and as well as transmission electron microscopy (TEM). The size was determined lower than 100 nm for synthetics. Then, a gel retardation assay was performed to investigate the entrapment efficiency of nano-complex. Afterward, the sort of in vitro assays was implemented on A549 (FA receptor-positive lung cancer cell line) and MRC5 (normal human diploid cell line) to evaluate the therapeutic efficiency of FA-CS-miR-126. Results: As the cell viability (40.7 ± 2.98% cell viability after 72 h treatment with 500 nM), migration assay (weaker migration after 24 h and 48 h), apoptotic and autophagy genes expression level (Caspse9: sixfolds; BAX: 17 folds; ATG5: fourfolds; and BECLIN1: threefolds more than the control group), the reduced expression level of EGF-L7, as a target gene for miR-126 was evaluated by Real-Time PCR too, then, cell cycle arrest (8.66% of cells in sub-G1 phase), and cell apoptosis assay (21.0% of cancer cell in late apoptosis phase) were scrutinized. Conclusion: These results are remarkably approved the biocompatible and efficient performance of FA-CS-miR-126 as a promising DDS.

The protective effects of Procyanidin C-1 on bisphenol a-induced testicular dysfunction in aged mice

The number and quality of sperm are decreased due to bisphenol A (BPA) exposure, an endocrine-disrupting chemical on the male reproductive system, especially in advanced paternal age (APA). Procyanidin C-1 (PCY-1), an antioxidant from grape seed (Vitis vinifera L.), has demonstrated anti-viral, anti-melanogenic and immunostimulatory effects. Therefore, this study aims to determine the effects of PCY-1 intervention on sperm parameters, testis morphological changes, serum testosterone, oestradiol, and luteinizing hormone (LH) concentrations and the expression of apoptotic (Bax and Bcl-2) and mitochondria-related (Mfn1 and Opa1) genes in BPA-exposed aged mice. Results revealed that PCY-1 intervention improves aged male fertility in BPA-exposed conditions by decreasing abnormal sperms percentage and increasing spermatogenic cell diameter and epithelial height. PCY-1 also decreased oestradiol, and increased LH and testosterone levels. The gene expression of Bax was significantly down-regulated by PCY-1 intervention. In contrast, Bcl-2 was substantially up-regulated. Expression of Mfn1 and Opa1 genes were also significantly up-regulated in the PCY intervention group. Hence, it is demonstrated that PCY-1 was able to mitigate the adverse effects of BPA on reproductive parameters of aged mice. Collectively, we postulated that PCY-1 has a potential role in protecting the ageing male reproductive system against the damaging impacts of BPA.

Impact of Group II Baculovirus IAPs on Virus-Induced Apoptosis in Insect Cells.

Apoptosis plays an important role in virus-host interactions and is a major element of the insect immune response. Exploring the regulatory mechanisms of virus-induced apoptosis through the expression of apoptotic genes holds important research and application value. Functional research on the reported inhibitor of apoptosis proteins (IAPs) mainly focuses on the group I baculovirus, while the functions of the group II baculovirus IAPs remains unclear. To explore its role in the regulation of the apoptosis of insect cells, we constructed the transient expression vector (pIE1 vectors) and the recombinant baculovirus expressing Bsiap genes (from the Buzura suppressaria nucleopolyhedrovirus) of the group II baculovirus. Apoptosis gene expression results and the virus-induced apoptosis rate show that the overexpression of BsIAP1 could promote apoptosis in insect cells. However, the overexpression of BsIAP2 and BsIAP3 decreases the expression of apoptotic genes, revealing an inhibitory effect. Results on the impact of baculovirus-induced apoptosis also confirm that BsIAP1 reduces viral nucleocapsid expression and the baculovirus titer, while BsIAP2 and BsIAP3 increase them significantly. Furthermore, compared with single expression, the co-expression of BsIAP2 and BsIAP3 significantly reduces the rate of virus-induced apoptosis and improves the expression of nucleocapsids and the titer of offspring virus, indicating the synergistic effect on BsIAP2 and BsIAP3. In addition, combined expression of all three BsIAPs significantly reduced levels of intracellular apoptosis-related genes (including apoptosis and anti-apoptosis genes), as well as apoptosis rate and progeny virus titer, indicating that life activities in insect cells are also inhibited. These findings reveal the relationship between apoptosis and group II baculovirus IAP, which provide an experimental and theoretical basis for further exploration of the molecular mechanism between group II baculoviruses and insect cells.