Expression Of Anti-apoptotic Gene Bcl-2 Research Articles

Comprehensive Chemical Profiling and Mechanistic Insight into Anticancer Activity of Annona muricata Leaves Extract.

The aqueous extract of Annona muricata L. leaves was thoroughly analyzed using the UPLC-MS/MS, in addition to a new approach of examination of the extract's impact on cancer of EAC(Ehrlich ascites carcinoma) in albino male mice. The aim was to investigate the diversity of the phytochemical constituents of the aqueous leaf capsule extract and their impacts on EAC as anticancer agents. The UPLC-ESI-MS/MS screening resulted in 410 tentatively identified metabolites. Among them, 384 compounds were tentatively identified in a previous study, besides a number of 26 compounds belonging to acetogenins, phenolics, flavonoids, alkaloids, and other miscellaneous compounds, which were exclusively identified in the aqueous extract of the leaf capsule. Interestingly, a new compound was tentatively characterized as galloyl-quinic acid-rutinoside. This study also demonstrated that treating EAC mice with an extract from A. muricata leaves significantly improved the abnormalities in the expression of pro-apoptotic (Bax and caspase-3) and anti-apoptotic (Bcl-2) genes. Furthermore, the extract showed good protection against induced Ehrlich hepatocarcinoma, according to the microscopical, histological, and immune-histochemical analyses of the liver tissues and tumor mass.

Evaluation of the cytotoxic activity of sorafenib-loaded camel milk casein nanoparticles against hepatocarcinoma cells.

Sorafenib, a multikinase inhibitor is used to treat hepatocellular and renal carcinoma. However, a low solubility impedes its bioavailability and thus, effectiveness. This study aims to enhance its effectiveness by using novel camel milk casein nanoparticles as a delivery system. This study evaluates the cytotoxicity of sorafenib encapsulated in camel milk casein nanoparticles against human hepatocarcinoma cells (HepG2 cells) in vitro. Optimal drug loaded nanoparticles were stable for 1 month, had encapsulation efficiency of 96%, exhibited a particle size of 230nm, zeta potential of -14.4 and poly disparity index of 0.261. Treatment with it led to cell morphology and DNA fragmentation as a characteristic of apoptosis. Flow cytometry showed G1 phase arrest of cell cycle and 26% increased apoptotic cells population upon treatment as compared to control. Sorafenib-loaded casein nanoparticles showed 6-fold increased ROS production in HepG2 cells as compared to 4-fold increase shown by the free drug. Gene and protein expression studies done by qPCR and western blotting depicted upregulation of tumor suppressor gene p53, pro-apoptotic Bax, and caspase-3 along with downregulated anti-apoptotic Bcl-2 gene and protein expression which further emphasized death by apoptosis. It is concluded regarding the feasibility of these casein nanoparticles as a delivery system with enhanced therapeutic outcomes against hepatocellular carcinoma cells.

Deoxycytidine kinase inactivation enhances gemcitabine resistance and sensitizes mitochondrial metabolism interference in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is considered one of the most lethal forms of cancer. Although in the last decade, an increase in 5-year patient survival has been observed, the mortality rate remains high. As a first-line treatment for PDAC, gemcitabine alone or in combination (gemcitabine plus paclitaxel) has been used; however, drug resistance to this regimen is a growing issue. In our previous study, we reported MYC/glutamine dependency as a therapeutic target in gemcitabine-resistant PDAC secondary to deoxycytidine kinase (DCK) inactivation. Moreover, enrichment of oxidative phosphorylation (OXPHOS)-associated genes was a common property shared by PDAC cell lines, and patient clinical samples coupled with low DCK expression was also demonstrated, which implicates DCK in cancer metabolism. In this article, we reveal that the expression of most genes encoding mitochondrial complexes is remarkably upregulated in PDAC patients with low DCK expression. The DCK-knockout (DCK KO) CFPAC-1 PDAC cell line model reiterated this observation. Particularly, OXPHOS was functionally enhanced in DCK KO cells as shown by a higher oxygen consumption rate and mitochondrial ATP production. Electron microscopic observations revealed abnormal mitochondrial morphology in DCK KO cells. Furthermore, DCK inactivation exhibited reactive oxygen species (ROS) reduction accompanied with ROS-scavenging gene activation, such as SOD1 and SOD2. SOD2 inhibition in DCK KO cells clearly induced cell growth suppression. In combination with increased anti-apoptotic gene BCL2 expression in DCK KO cells, we finally reveal that venetoclax and a mitochondrial complex I inhibitor are therapeutically efficacious for DCK-inactivated CFPAC-1 cells in in vitro and xenograft models. Hence, our work provides insight into inhibition of mitochondrial metabolism as a novel therapeutic approach to overcome DCK inactivation-mediated gemcitabine resistance in PDAC patient treatment.

Anti-cancer effects of green synthesized gold nanoparticles using leaf extract of Annona muricata. L against squamous cell carcinoma cell line 15 through apoptotic pathway

Background: Oral cancer remains one of the most dreadful diseases in developing nations. Currently, there has been a rise in the prevalence of tongue squamous cell carcinoma (SCC), with a poor prognosis. The use of standard treatment approaches against oral cancer patients brings about several side effects. In recent years, nanomedicine has provided a versatile platform for developing new targeted therapeutic modalities. However, safety remains a concern in the synthesis of nanoparticles (NPs). Therefore, the present study aims to synthesize safer phytoconstituent-mediated gold NPs (AuNPs) utilizing leaf extracts of Annona muricata, where the biochemical components of the plant leaf act as the reducing and capping agents in the synthesis of NPs, and to evaluate its anti-cancer activity against SCC. Materials and Methods: In this in vitro experimental study, AuNPs were synthesized through an effective, simple, and ecologically sound green synthesis method. After characterization of these synthesized AuNPs, in vitro assays such as 3-(4, 5-dimethylthiazole2-yl)-2, 5-biphenyl tetrazolium bromide, wound healing, and clonogenic assays were carried out to investigate the anti-cancer potential of green synthesized AuNPs in the human tongue SCC cell line (SCC-15), and the possible mechanism of action was evaluated through gene and protein expression analysis of Bax, Bcl-2, and p53 genes. The results were expressed as mean ± standard deviation using Statistical Package for Social Sciences (SPSS) 20.0 software and Student's t-test was performed for experimental data. P ≤0.05 were considered statistically significant. Results: The in vitro assays demonstrated that the synthesized AuNPs are exhibiting anti-cancer activity by apoptosis of SCC-15 cells in a dose-dependent manner. Further, it also revealed a highly significant decrease in anti-apoptotic Bcl-2 gene expression, whereas pro-apoptotic genes p53 and Bax revealed a highly significant increase, which is statistically significant compared to the control (P < 0.05). Conclusion: Our findings demonstrated that the AuNPs synthesized from A. muricata leaf extract could act as a novel anticancer agent, particularly against SCC, after further scrutiny.

Designing nanostructured lipid carriers modified with folate-conjugated chitosan for targeted delivery of osthole to HT-29 colon cancer cells: investigation of anticancer, antioxidant, and antibacterial activities

AbstractThe present study proposed to design nanostructured lipid carriers (NLC) coated with chitosan (CS) conjugated folate (FA) for the targeted delivery of Osthole (OST) to the HT-29 colon cancer cell line and improve its anticancer capability. To assess the physicochemical characteristics of OST-loaded NLC decorated with CS-conjugated FA (OST-NCF-NPS), several techniques, including DLS, SEM, and FTIR, were applied. After determining the encapsulation efficiency of OST in CSFA-modified NLC-NPs, an MTT test was conducted to evaluate the cytotoxic effects of this nano platform on the HT-29 cancer cell line in comparison to normal HFF cells. Possible mechanisms of apoptosis in cancer cells treated with OST-NCF-NPs were examined using qPCR, flow cytometry, and AO/PI fluorescent staining methods. Moreover, the antioxidant capacity of these biosynthesized nanocarriers was determined using ABTS and DPPH methods, and their antibacterial potential was measured through disk diffusion, MIC, and MBC assays. According to the findings, OST-NCF-NPS had the ideal average size of 179.19 nm, low polydispersity (PI = 0.23), acceptable physical stability (ζ-potential = + 18.99 mV), and high entrapment efficiency (83.5%). The MTT data demonstrated the selective cytotoxicity of NPs toward cancerous cells compared to normal cells. Cell cycle and Annexin V/Propidium Iodide (AnV/PI) analysis indicated that OST-NCF-NPs increased the sub-G1 population and AnV/PI-positive cells. The occurrence of programmed cell death in the treated cells was also verified by altered expression of proapoptotic (BAX and caspase-3) and antiapoptotic (Bcl-2) genes. Furthermore, the NPs exhibited strong antibacterial activity, particularly against gram-negative bacteria, and high antioxidant effects in reducing ABTS and DPPH-free radicals. Graphical Abstract

Expression of Apoptosis and Autophagy Genes in HeLa and HEK 293 Cells under Conditions of Nutrient Deprivation

The goal of the study was a comparing the degree of development of autophagy in the human cervical carcinoma cells of HeLa-V and HeLa-R sublines and non-tumor human embryonic kidney cells HEK 293 under two types of starvation conditions – 24- and 48-h culture in serum-free DMEM medium and 4-h incubation in Earle’s minimal medium. The work assessed cell viability using MTT method and the expression of apoptosis (BCL2, BAX, CASP3) and autophagy (ULK1, BECN1, ATG5, ATG14, MAP1LC3B) genes using real-time PCR. Cultivation under serum starvation and Earl’s medium resulted in a significant decrease in the viability of HEK 293 cells, but had no influence on HeLa-V and HeLa-R cells. In the tumor cells of both lines, the expression of anti-apoptotic gene BCL2 increased, while in HEK 293 cells the BCL2/BAX ratio decreased and CASP3 gene was activated. In HeLa-V and HeLa-R cells, nutrient deprivation induced the stimulation of various combinations of genes ULK1, BECN1, ATG5 and ATG14 implicated in the initial stages of autophagy, but none of the treatments affected the expression of MAP1LC3B gene. In HEK 293 cells, serum starvation led to increase in expression level of BECN1, ATG5, ATG14 and MAP1LC3B genes. Thus, stimulation of autophagy in HeLa cells, especially HeLa-R, prevents the development of apoptotic processes, while in HEK 293 cells the processes of apoptosis and autophagy occur in parallel. Culture in the serum-free DMEM for 48 h appears to be most effective way to induce autophagy in tumor cell lines and, accordingly, the most suitable model for studying the role of autophagy in the development of their resistance to apoptotic pathway of death.

Helicteres isora Ethanol Extract Induces Apoptotic Cell Death in HepG2 Cells

Background: H. isora is a species of the genus Helicteres, a medicinal plant known to be used in the treatment of liver cancer in Vietnam. However, there has been no recorded about biological activity of H. isora species in Vietnam. The current study aimed to study the antitumor activity through its antioxidant activity and inhibitory properties on hepatocellular carcinoma cells. Methods: Arial parts of Helicteres isora were collected from Dak Nong province. Samples were extracted maceration method in ethanol solvent. The antitumor activity were identified though the DPPH radical scavenging activity (Brand-Williams et al., (1995) and WST-1 assay, Real- time PCR, Hoechst 33258 spectrofluorometric assay and Annexin V-FITC Apoptosis Assay on the human liver cancer cells (HepG2) and non-malignant cells of Chang’s Live. Result: The ethanol extract of H. isora exhibited antioxidant activity with IC50=21.16 µg/ml. H. isora extract also exhibited inhibitory activity on HepG2 cell division with IC50=37 µg/ml after 48 h. The expression of DNA fragmentation of the extract also increased with time (48-72h). Apoptosis induction of extract was associated with up-regulation of pro-apoptotic Bax gene expression and down-regulation of anti-apoptotic Bcl-2 gene expression. The H. isora extract was found to induce the apoptotic process in HepG2 cells through the up and down regulation of the Bax/Bcl-2 gene.

Antioxioxidant and antiapoptotic effects of Thymosin β4 in Aβ-induced SH-SY5Y cells via the 5-HTR1A/ERK axis.

Alzheimer's disease (AD) is a common amnestic cognitive impairment characterised by β-amyloid (Aβ) plaques deposit in the brain of the elderly. AD is a yet incurable disease due to its unknown exact pathogenesis and unavailability of effective remedies in clinical application. Thymosin β4 (Tβ4) is a housekeeping protein that plays important role in cell proliferation, migration and differentiation. It has the ability to protect and repair neurons however it is still unclear involvement in AD. Therefore, the aim of this study is to elucidate the role and mechanism of Tβ4 in mediating the improvement of AD. AD-like cell model was constructed in neuroblastoma cell line SH-SY5Y treated with Aβ. Overexpression of Tβ4 were done using lentivirus infection and downregulation through siRNA transfection. We performed western blot and flow cytometry to study the apoptosis and standard kits to measure the oxidative stress-associated biomarkers. There is significant increased in viability and decreased apoptosis in Tβ4 overexpression group compared to control. Furthermore, overexpression of Tβ4 suppressed the expression of pro-apoptotic markers such as Caspase-3, Caspase-8, and Bax meanwhile upregulated the expression of anti-apoptotic gene Bcl-2. Tβ4 alleviated oxidative damage by reducing MDA, LDH and ROS and increasing SOD and GSH-PX in Aβ-treated SH-SY5Y cells. We found that Tβ4 inhibit ERK/p38 MAPK pathway and intensify the expression of 5-HTR1A. Additionally, we showed that upregulation of 5-HTR1A dampened the Tβ4 to activate ERK signalling. In conclusion, our study revealed the neuroprotective role of Tβ4 in AD which may open up new therapeutic applications in AD treatment.

Effects of niacin supplementation during in vitro culture on the developmental competence of porcine embryos.

Niacin is a water-soluble vitamin belonging to the vitamin B complex. It has been found to possess various biological activities, including antioxidant and lipid modification capacities. This study aimed to elucidate the effects of niacin treatment in porcine in vitro culture (IVC) medium on embryo developmental competence after parthenogenetic activation. IVC medium was supplemented with different concentrations of niacin (0 [control], 300, 600 and 900 μM). The results showed that embryos cultured in an IVC medium supplemented with 300 and 600 μM niacin had an increased cleavage rate (p < .05). In addition, 300 μM niacin treatment resulted in a higher blastocyst formation rate than the control and other niacin-treated groups. However, the total cell number did not differ significantly among the experimental groups. Niacin supplementation at 600 μM decreased reactive oxygen species, whereas treatment with 300, 600 and 900 μM increased glutathione levels in day two embryos. On day seven, 300 μM niacin exhibited improved fatty acid levels and fewer lipid droplets than the control group. Furthermore, gene expression at the mRNA level was performed on day two and day seven embryos, treated with or without 300 μM niacin. The expression of anti-apoptotic BCL2 and lipid metabolism PLIN2-related genes were upregulated, whereas the pro-apoptotic BAX and CASPASE3 were downregulated with niacin supplementation compared with the control group. However, SIRT1, a gene related to energy and the oxidative state, was up-regulated in niacin-treated day two embryos (p < .05). Overall, the results indicate that niacin has a beneficial effect on pre-implantation embryo development by modulating lipid metabolism and reducing oxidative stress and apoptosis. The expression patterns of PLIN2 and SIRT1 reported here suggest that these transcripts may be involved in the mechanism by which niacin affects the developmental capacity of IVC embryos.

Studying Luminal, A and B subtypes of the breast cancer under paracrine secretion of fibroblasts

Introduction Understanding the role of the tumor microenvironment in specifying molecule markers of breast cancer subtypes could involve tumor microenvironment properties in subtyping and clinical assessment of breast cancer. Therefore, interconversion of luminal states and or their specific markers alteration under the control of TME particularly CAFs deserve to be more investigated. Methods To activate normal human fibroblasts, liquid overlay technique or nemesis was used and α-SMA expression, CAFs marker, in fibroblastic spheroids was measured by blotting. The luminal A, MCF-7, and luminal B, MDA-MB 361, cell lines are treated with normal and spheroidal/activated fibroblast conditioned medium for 48 hours. The morphological changes of luminal A and B cells were evaluated by invert microscopy and analyzed by the shape factor formula. Moreover, sensitivity changes to chemotherapeutic agents, Ki67 expression, and gene expression level including ER-related and proliferative genes were assessed respectively by MTT assay, Immunofluorescence, and real time qRT-PCR methods. Results Activated fibroblast, spheroid form, by LOT/nemosis expressed α-SMA marker two folds more than monolayer cultured fibroblasts. Our study underlined a significant increase in IC50 of both luminal A and B cell lines after being co-cultured with conditioned medium markedly in spheroidal conditioned medium. Morphological changes study by shape factor formula demonstrated more aggressiveness with gaining mesenchymal features both in luminal A and B subtypes by increasing exposure time. Changes in Ki67 expression level were observed after fibroblasts and spheroidal paracrine conditioning. Only this data supports the luminal interconversion by elevated Ki67 expression in luminal A and lowered Ki67 expression in luminal B. Gene expression results revealed that the MCF7 and MDA-MB cells as representatives luminal A and B subtypes in this study did not interchange their own specific gene pattern, however both luminal types in co-culture with condition medium increased the expression of anti-apoptotic Bcl2 gene. Conclusion Under the paracrine influence of fibroblast cells, both luminal A(MCF7) and B (MDA-MB) subtypes of breast cancer gained invasive, anti-apoptotic, and chemoresistance features which are mostly increased by activated fibroblast conditioned medium mimicking CAFs. There was no strong proof for interconversion of luminal A and B, with more similarities, subtypes of breast cancer.

Expression of Apoptosis-Related Genes in Cultured Ovarian Tissue of Mouse in Presence of Coenzyme Q10.

Oxidative stress affects the development of ovarian follicles during in vitro culture thus applying an antioxidant is necessary. The aim of this study was to investigate the effect of coenzyme Q10 (CoQ10) on the expression of apoptosis-related genes of mouse ovaries during in vitro culture. The immature mouse ovaries were cultured in the presence of 50 μM CoQ10 for 7 days. Histological examinations were performed and the 17 beta-estradiol concentration was measured on the seventh day of culture. The relative expression of Caspase 3, Bax, Bad, and Bcl 2 genes were investigated by real-time RT-PCR. The rates of normal follicles in the presence of CoQ10 was significantly increased compared to the control group. Also, in CoQ10-trated group a significant increase in the level of 17 beta-estradiol was seen compared to the control group. The mRNA expression of anti-apoptotic gene Bcl2 was significantly increased while the expression of pro-apoptotic genes (Caspase3, Bax and Bad) significantly declined in CoQ10 treated group compared to those of control group. The supplementation of the ovarian culture media with CoQ10 improved the follicular development through alteration in expression of apoptosis-related genes and stimulated the production of estradiol.

Increased Anticancer Activity of Organic Kimchi with Starters Demonstrated in HT-29 Cancer Cells

This study aims to investigate the effects of kimchi made with organic ingredients and lactic acid bacteria (LAB) as starters (Leuconostoc mesenteroides + Lactiplantibacillus plantarum) on HT-29 human colon carcinoma cells. Four types of kimchi (standard kimchi (SK), commercial kimchi (CK), anticancer kimchi (AK), and organic anticancer kimchi (OAK)) were evaluated. The results show that, among the different types of kimchi studied, OAK presents high DPPH free-radical scavenging activity and total phenol and flavonoid contents, and the MTT assay shows that the growth inhibition rate against HT-29 cancer cells is the highest. In addition, the quantitative reverse transcription polymerase chain reaction (RT-qPCR) results show that, compared to SK and CK, AK and OAK can effectively down-regulate the mRNA expression of anti-apoptotic gene Bcl-2 and up-regulate the mRNA expression of the cell cycle arrest genes p21 and p53; pro-apoptotic genes Bim, Bak, and Bad; and genes for caspases 3 9. Subsequently, a Western blot test confirmed that the expression of Bcl-2 decreased and the expressions of p53, Bax, and caspases 3 and 9 increased in OAK. The abovementioned results indicate that the anticancer kimchi prepared with organic ingredients and starters of lactic acid bacteria effectively present the best antioxidant activity and inhibit the proliferation of HT-29 cancer cells by promoting apoptosis and cell cycle arrest.

EIF2α incites photoreceptor cell and retina damage by all-trans-retinal

Dry age-related macular degeneration (AMD) and recessive Stargardt's disease (STGD1) lead to irreversible blindness in humans. The accumulation of all-trans-retinal (atRAL) induced by chaos in visual cycle is closely associated with retinal atrophy in dry AMD and STGD1 but its critical downstream signaling molecules remain ambiguous. Here, we reported that activation of eukaryotic translation initiation factor 2α (eIF2α) by atRAL promoted retinal degeneration and photoreceptor loss through activating c-Jun N-terminal kinase (JNK) signaling-dependent apoptosis and gasdermin E (GSDME)-mediated pyroptosis. We determined that eIF2α activation by atRAL in photoreceptor cells resulted from endoplasmic reticulum homeostasis disruption caused at least in part by reactive oxygen species production, and it activated JNK signaling independent of and dependent on activating transcription factor 4 and the activating transcription factor 4/transcription factor C/EBP homologous protein (CHOP) axis. CHOP overexpression induced apoptosis of atRAL-loaded photoreceptor cells through activating JNK signaling rather than inhibiting the expression of antiapoptotic gene Bcl2. JNK activation by eIF2α facilitated photoreceptor cell apoptosis caused by atRAL via caspase-3 activation and DNA damage. Additionally, we demonstrated that eIF2α was activated in neural retina of light-exposed Abca4-/-Rdh8-/- mice, a model that shows severe defects in atRAL clearance and displays primary features of human dry AMD and STGD1. Of note, inhibition of eIF2α activation by salubrinal effectively ameliorated retinal degeneration and photoreceptor apoptosis in Abca4-/-Rdh8-/- mice upon light exposure. The results of this study suggest that eIF2α is an important target to develop drug therapies for the treatment of dry AMD and STGD1.

Wound Healing Activity of the Flavonoid-Enriched Fraction of Selaginella bryopteris Linn. against Streptozocin-Induced Diabetes in Rats

Diabetes and its complications, such as delayed wound healing, are increasing at an alarming rate in India, putting an enormous strain on the country’s limited healthcare resources. Hence, the present study proposes to screen/identify the possible mechanisms and to study the effect of the flavonoid-enriched fraction of Selaginella bryopteris extract against human keratinocyte cell lines (HaCaT) and streptozocin (STZ)-induced diabetic wounds in a male Wistar rat model. Chemical profiling was performed by an MTT assay. The obtained GC–MS analysis results showed the presence of amentoflavone, gallic acid, imidazole, palmitic acid, catechine, L-fucitol, lupeol, and myo-inositol as the major bioactive phytoconstituents. S. bryopteris induces the generation of ROS, the condensation of chromatin in the nucleus, and changes in the membrane potential of mitochondria in HaCaT cell lines. An S. bryopteris-dependent induction of apoptosis-mediated cell death in HaCaT cell lines was confirmed by an AO/PI analysis. Mitochondrial depolarization was reflected in JC-1 staining of cells. The wound size was reduced and epithelialization was enhanced. Keratinocyte migration decreased interleukins, TNF-α, IL-2, and IL-6 and the expression of pro-apoptotic (p53, caspase-3, caspase-9, and Bax) and anti-apoptotic (Bcl-2) genes in a dose-dependent manner. Keratinocyte migration increased antioxidant enzyme levels (CAT, SOD, MDA, and GSH). Wound healing is facilitated through the mitochondria-mediated apoptosis pathway, revealing a new area of diabetic wound therapy.

Effect of reactive oxygen species in cadmium chloride induced apoptosis of mouse Leydig cells

To study the effect of reactive oxygen species(ROS) in cadmium chloride-induced apoptosis of mouse Leydig cells(TM3 cells) and explore the underlying molecular mechanisms.TM3 cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 μmol/L) for 24 h. CCK-8 assay was used to detect the effect of CdCl_2 on TM3 cell activity. Hoechst33342 staining was performed to explore the formation of apoptotic bodies. DCFH-DA probe was used to detect the level of ROS in the cells. TM3 cells were pretreated with 1 mmol/L NAC for 1 h and then treated with 10 μmol/L CdCl_2 for 24 h. The protein expression levels of pro-apoptotic proteins Caspase-9 and cleaved Caspase-3 were detected by Western blot; RT-qPCR was used to measure the expression of anti-apoptotic gene Bcl-2 and pro-apoptotic genes Caspase-9 and Caspase-3.After exposure to CdCl_2 for 24 h, viability of TM3 cells decreased and the number of apoptotic bodies increased. Western blot result showed that the protein level of Caspase-9 in the 10 μmol/L CdCl_2 treatment group was increased to 0.86±0.10(P&lt;0.05) compared with the control group(0.56±0.07). Compared with the control group(0.37±0.11), the protein level of cleaved Caspase-3 in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 0.65±0.03 and 1.05±0.13(P&lt;0.05). Compared with the control group(46.80±1.24), the intracellular ROS content in the 5 and 10 μmol/L treatment groups increased to 60.47±1.39 and 80.63±1.34(P&lt;0.05). Compared with the cadmium-treated group, NAC inhibited Caspase-9(CdCl_2 group: 0.89±0.07; CdCl_2+NAC group: 0.28±0.02)and cleaved Caspase-3(CdCl_2 group: 1.53±0.21; CdCl_2+NAC group: 0.66 ±0.07), the difference was statistically significant(P&lt;0.05). At the same time, NAC decreased the ROS level(62.64±0.93) in the CdCl_2 exposure group(80.13±0.94)(P&lt;0.05). In addition, RT-qPCR result showed that the Caspase-9 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were 1.40±0.14 and 1.90±0.12(P&lt;0.05), compared with the control group(0.97±0.10). Compared with the control group(0.88±0.08), the cleaved Caspase-3 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 1.42±0.11 and 1.59±0.12(P&lt;0.05). While in the 5 and 10 μmol/L CdCl_2-treated group, compared with the control group(0.94±0.02), the Bcl-2 mRNA level were decreased to 0.60±0.02 and 0.50±0.09(P&lt;0.05). Compared with the cadmium treatment group(0.57±0.06), NAC could significantly improve the cadmium-induced Bcl-2 mRNA expression level(0.92±0.03), and Caspase-9(CdCl_2 group: 1.96±0.07; CdCl_2+NAC group: 1.04±0.02) and Caspase-3(CdCl_2 group: 1.65±0.02; CdCl_2+NAC group: 0.66±0.04) were decreased(P&lt;0.05).The Caspase cascade in mouse Leydig cells can be activated by excessive ROS induced by CdCl_2, and inhibition of ROS production can significantly reduce the CdCl_2-induced apoptosis of TM3 cells.

Effect of reactive oxygen species in cadmium chloride induced apoptosis of mouse Leydig cells

To study the effect of reactive oxygen species(ROS) in cadmium chloride-induced apoptosis of mouse Leydig cells(TM3 cells) and explore the underlying molecular mechanisms. TM3 cells were used as an in vitro model for studying reproductive toxicity induced by cadmium exposure. The cells were treated with different concentrations of CdCl_2(0, 5 and 10 μmol/L) for 24 h. CCK-8 assay was used to detect the effect of CdCl_2 on TM3 cell activity. Hoechst33342 staining was performed to explore the formation of apoptotic bodies. DCFH-DA probe was used to detect the level of ROS in the cells. TM3 cells were pretreated with 1 mmol/L NAC for 1 h and then treated with 10 μmol/L CdCl_2 for 24 h. The protein expression levels of pro-apoptotic proteins Caspase-9 and cleaved Caspase-3 were detected by Western blot; RT-qPCR was used to measure the expression of anti-apoptotic gene Bcl-2 and pro-apoptotic genes Caspase-9 and Caspase-3. After exposure to CdCl_2 for 24 h, viability of TM3 cells decreased and the number of apoptotic bodies increased. Western blot result showed that the protein level of Caspase-9 in the 10 μmol/L CdCl_2 treatment group was increased to 0.86±0.10(P&lt;0.05) compared with the control group(0.56±0.07). Compared with the control group(0.37±0.11), the protein level of cleaved Caspase-3 in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 0.65±0.03 and 1.05±0.13(P&lt;0.05). Compared with the control group(46.80±1.24), the intracellular ROS content in the 5 and 10 μmol/L treatment groups increased to 60.47±1.39 and 80.63±1.34(P&lt;0.05). Compared with the cadmium-treated group, NAC inhibited Caspase-9(CdCl_2 group: 0.89±0.07; CdCl_2+NAC group: 0.28±0.02)and cleaved Caspase-3(CdCl_2 group: 1.53±0.21; CdCl_2+NAC group: 0.66 ±0.07), the difference was statistically significant(P&lt;0.05). At the same time, NAC decreased the ROS level(62.64±0.93) in the CdCl_2 exposure group(80.13±0.94)(P&lt;0.05). In addition, RT-qPCR result showed that the Caspase-9 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were 1.40±0.14 and 1.90±0.12(P&lt;0.05), compared with the control group(0.97±0.10). Compared with the control group(0.88±0.08), the cleaved Caspase-3 mRNA levels in the 5 and 10 μmol/L CdCl_2 treatment groups were increased to 1.42±0.11 and 1.59±0.12(P&lt;0.05). While in the 5 and 10 μmol/L CdCl_2-treated group, compared with the control group(0.94±0.02), the Bcl-2 mRNA level were decreased to 0.60±0.02 and 0.50±0.09(P&lt;0.05). Compared with the cadmium treatment group(0.57±0.06), NAC could significantly improve the cadmium-induced Bcl-2 mRNA expression level(0.92±0.03), and Caspase-9(CdCl_2 group: 1.96±0.07; CdCl_2+NAC group: 1.04±0.02) and Caspase-3(CdCl_2 group: 1.65±0.02; CdCl_2+NAC group: 0.66±0.04) were decreased(P&lt;0.05). The Caspase cascade in mouse Leydig cells can be activated by excessive ROS induced by CdCl_2, and inhibition of ROS production can significantly reduce the CdCl_2-induced apoptosis of TM3 cells.

Lutein combined with EGCG improved retinitis pigmentosa against N-methyl-N nitrosourea-induced.

In order to investigate the synergistic improving effect of lutein (LUT) and epigallocatechin-3-gallate (EGCG) treatment on retinitis pigmentosa (RP), an N-methyl-N-nitrosourea (MNU)-induced mouse model was conducted in the present study. Compared to the LUT alone treatment group, in the LUT combined with EGCG (LUT-EGCG) treatment group, the accumulation content of LUT was significantly increased by 50.24% in the liver. The morphological results indicated that LUT-EGCG treatment significantly improved the retina structure with the thickness of the outer nuclear layer restored to 185.28 ± 0.29 μm, showing no significant difference compared to the control group. The LUT-EGCG treatment also increased the production of short-chain fatty acids, such as acetic and propionic acids. Compared with the LUT alone treatment, the LUT-EGCG treatment significantly increased the relative abundance of Lachnospiraceae and Helicobacteraceae. RT-qPCR results indicated that LUT-EGCG treatment significantly increased the antiapoptotic gene Bcl-2 expression. In addition, the expression of IL-6 was significantly down-regulated in the LUT-EGCG group, while there was no significance in NF-κβ, TNF-α, IL-1β, and IL-18 compared with the LUT group. Correlation analysis supported the conclusion that LUT combined with EGCG may improve RP by modulating antiapoptotic gene expression and regulating the abundance of gut microbiota. However, the underlying mechanism still needs further research.

Dietary flavonoid carvacrol triggers the apoptosis of human breast cancer MCF-7 cells via the p53/Bax/Bcl-2 axis

Recently, numerous studies have shown that natural compounds such as carvacrol can be used as therapeutic agents for medicinal purposes. Although carvacrol was found to suppress breast cancer cell lines, however, whether the therapeutic effect of carvacrol on breast cancer is caused by increasing the expression of pro-apoptotic genes in the MCF-7 cell line has not been studied. In this research, we investigate the effect of carvacrol on the expression of P53, pro-apoptotic Bax and anti-apoptotic BCL-2 genes in MCF-7 cells. After preparation and cultivation of MCF-7 cells, the IC50 value of carvacrol on the cells was evaluated by MTT assay, and then apoptosis induction was observed in the cell line treated with different concentrations of carvacrol by DAPI staining. To assess the expression level of Bax, P53 and Bcl-2 both in genes and protein levels QPCR method and western blot analysis were used. According to the results of the research, it was determined that the IC50 of carvacrol compound in MCF-7 cells is 305µM. DAPI staining exhibit apoptosis and morphological changes in treated MCF-7 cells. Real-time PCR assay and western blot showed increasing the Bax and P53 expression and decreasing the expression of the Bcl-2 anti-apoptotic gene and protein. These results showed that carvacrol has cell growth inhibition effects on the MCF-7 cancer cell line. Carvacrol-induced p53-dependent apoptosis, which might be related to the Bax/Bcl-2 associated pathway. These results indicated that carvacrol could be considered suitable for the prevention and treatment of breast cancer.

Induction of ROS mediated genomic instability, apoptosis and G0/G1 cell cycle arrest by erbium oxide nanoparticles in human hepatic Hep-G2 cancer cells

The remarkable physical and chemical characteristics of noble metal nanoparticles, such as high surface-to-volume ratio, broad optical properties, ease of assembly, surfactant and functional chemistry, have increased scientific interest in using erbium oxide nanoparticles (Er2O3-NPs) and other noble metal nanostructures in cancer treatment. However, the therapeutic effect of Er2O3-NPs on hepatic cancer cells has not been studied. Therefore, the current study was conducted to estimate the therapeutic potential of Er2O3-NPs on human hepatocellular carcinoma (Hep-G2) cells. Exposure to Er2O3-NPs for 72 h inhibited growth and caused death of Hep-G2 cells in a concentration dependent manner. High DNA damage and extra-production of intracellular reactive oxygen species (ROS) were induced by Er2O3-NPs in Hep-G2 cells. As determined by flow cytometry, Er2O3-NPs arrested Hep-G2 cell cycle at the G0/G1 phase and markedly increased the number of Hep-G2 cells in the apoptotic and necrotic phases. Moreover, Er2O3-NPs caused simultaneous marked increases in expression levels of apoptotic (p53 and Bax) genes and decreased level of anti-apoptotic Bcl2 gene expression level in Hep-G2 cells. Thus it is concluded that Er2O3-NPs inhibit proliferation and trigger apoptosis of Hep-G2 cells through the extra ROS generation causing high DNA damage induction and alterations of apoptotic genes. Thus it is recommended that further in vitro and in vivo studies be carried out to study the possibility of using Er2O3-NPs in the treatment of cancer.

Magnesium yields opposite effects on the nuclear and cytosolic cascades of apoptosis in different rat brain regions.

Magnesium is considered as potential neuroprotective and therapeutic agent, but certain studies have provided evidence of its apoptotic effectiveness in neurons. We aimed to evaluate the possible apoptotic effects of long-term magnesium use in healthy adult rat brains. Magnesium citrate and magnesium glycinate compounds were administered orally to rats for 8 weeks (36 mg/kg). Expression levels of Bcl-2, Bax and Cyt-C genes were analyzed by real-time polymerase chain reactions (RT-PCR) in the prefrontal cortex, hippocampus and striatum regions. Bcl-2, Bax and CytC protein levels were measured using ELISA kits. Tissue sections were evaluated histopathologically with hematoxylin-eosin staining. Compared to the control group, the magnesium-administered groups indicated gene expression reductions in almost all brain regions; pro-apoptotic Bax, anti-apoptotic Bcl-2 and Cyt-C gene expression levels were reduced. With magnesium, the Bcl-2 and Bax protein levels were increased. Bax/Bcl-2 gene and protein ratio were also increased in the striatum and hippocampus, whereas Cyt-C protein levels were decreased or did not change in the magnesium treated groups. There was no pathological finding in histological evaluation. Long-term magnesium usage can promote apoptotic cascade in brain tissue by increasing Bax/Bcl-2 ratio. Cyt-C, a prominent factor processing caspase pathway, was decreased or unchanged. In addition, taking into account the histological evaluation, we supposed that the absence of Cyt-C in the cytosol can prevent the subsequent apoptotic pathway. Consequently, we obtained the findings of apoptotic initiation with magnesium in brain, but this cascade seems to be arrested at later stages.