Expression Of Anti-apoptotic Gene Bcl-2 Research Articles

Thiacloprid Exposure Induces Oxidative Stress, Endoplasmic Reticulum Stress, and Apoptosis in the Liver of Mauremys reevesii.

Among neonicotinoid insecticides, thiacloprid (THI) is extensively utilized in agricultural practices, which poses a potential toxicity risk to aquatic fauna. Turtles, integral to aquatic ecosystems, have not yet been comprehensively assessed for their vulnerability to THI exposure. In this study, we aimed to evaluate the effects of THI on oxidative stress, endoplasmic reticulum stress (ERS), and apoptosis in aquatic turtles. We categorized Mauremys reevesii into three groups: a control group and two experimental groups exposed to environmentally relevant (4.5 μg/mL) and high (15 mg/mL) concentrations of THI, respectively. Transcriptome analysis revealed that genes significantly associated with the elimination of superoxide radicals, organelle inner membrane functions, peroxiredoxin activity, and apoptotic pathways were abundantly expressed in the high-concentration THI group. Notably, exposure to high concentrations of THI led to a marked increase in glutathione peroxidase (GPX) and superoxide dismutase (SOD) activities, whereas catalase (CAT) activity declined and malondialdehyde (MDA) levels rose, indicating the presence of oxidative stress. Moreover, THI upregulated the expression of the ER stress marker GRP78. Simultaneously, the mRNA levels of pivotal unfolded protein response genes, including AFT6, AFT4, IRE1α, CHOP, XBP1, and eIF2α, were significantly elevated in response to THI exposure. Furthermore, high concentrations of THI significantly activated the activities of caspase-3, caspase-8, and caspase-9 enzymes in the liver tissue. The expression of anti-apoptotic gene Bcl-2 was downregulated, whereas the pro-apoptotic genes Bax and caspase-3 were upregulated, leading to an increase in hepatic apoptotic cells following THI exposure. Collectively, our study indicates that THI can induce hepatic damage in turtles through the promotion of oxidative stress, ERS, and apoptosis. These findings gain a deeper understanding of the toxic effects of THI on keystone species in aquatic ecosystems, thereby improving our overall understanding of their environmental impacts.

Calcium hydroxide nanoparticles induce cell death, genomic instability, oxidative stress and apoptotic gene dysregulation on human HepG2 cells

Calcium hydroxide nanoparticles (Ca(OH)2NPs) possess potent antimicrobial activities and unique physical and chemical properties, making them valuable across various fields. However, limited information exists regarding their effects on genomic DNA integrity and their potential to induce apoptosis in normal and cancerous human cell lines. This study thus aimed to evaluate the impact of Ca(OH)2NPs on cell viability, genomic DNA integrity, and oxidative stress induction in human normal skin fibroblasts (HSF) and cancerous hepatic (HepG2) cells. Cell viability and genomic DNA stability were assessed using the Sulforhodamine B (SRB) assay and alkaline comet assay, respectively. Reactive oxygen species (ROS) levels were measured using 2,7-dichlorofluorescein diacetate, while the expression level of apoptosis-related genes (p53, Bax, and Bcl-2) were quantified using real-time PCR (qRT-PCR). The SRB cytotoxicity assay revealed that a 48-hour exposure to Ca(OH)2NPs caused concentration-dependent cell death and proliferation inhibition in both HSF and HepG2 cells, with IC50 values of 271.93 µg/mL for HSF and 291.8 µg/mL for HepG2 cells. Treatment with the IC50 concentration of Ca(OH)2NPs selectively induced significant DNA damage, excessive ROS generation, and marked dysregulation of apoptotic (p53 and Bax) and anti-apoptotic (Bcl-2) gene expression in HepG2 cells, triggering apoptosis. In contrast, exposure of HSF cells to the IC50 concentration of Ca(OH)2NPs caused no significant changes in genomic DNA integrity, ROS generation, or apoptotic gene expression. These findings indicate that Ca(OH)2NPs exhibit concentration-dependent cytotoxicity in both normal HSF and cancerous HepG2 cells. However, exposure to the IC50 concentration was non-genotoxic to normal HSF cells while selectively inducing genotoxicity and apoptosis in HepG2 cancer cells through DNA breaks and ROS-mediated mechanisms. Further studies are required to explore the biological and toxicological properties and therapeutic potential of Ca(OH)2NPs in hepatic cancer treatment.

Unraveling the Ferroptosis-inducing Potential of Methanol Leaves Extract of Prosopis Juliflora Via Downregulation of SLC7A11 and GPX4 mRNA Expression in A549 Lung Cancer Cells.

Prosopis juliflora has been employed in many traditional treatments. As evidenced by our earlier research, Prosopis juliflora leaf methanol extract (PJME) has a promising future in the fight against lung cancer. It may also be used in conjunction with other treatments to effectively manage lung cancer. The main objective of this study was to explore the potential of PJME to inhibit lung cancer in A549 cells, along with its underlying mechanisms of action. The antiproliferative effects were determined using MTT and LDH tests. Apoptosis- inducing capacity was evaluated using the DAPI staining, caspase-3 test, cytochrome C assay, PARP cleavage, and qRT-PCR. To investigate the mechanism of action of PJME in lung cancer, the levels of ROS, MMP, GSH, MDA, and specific ferroptosis indicators were measured. The experimental data of the current study indicated that exposure of A549 cells to PJME reduced cell viability and increased cellular cytotoxicity. The apoptosis-inducing ability of PJME in A549 cells was validated by enhanced nuclear condensation, level of the caspase- 3, cytochrome C, and PARP release. In addition, qRT-PCR investigations verified that the administration of PJME led to a decrease in the expression of anti-apoptotic gene Bcl2 while enhancing the mRNA level of pro-apoptotic genes, such as Bax and caspase-3, in A549 cells. The study also found that PJME has the ability to activate ferroptosis pathways, as evidenced by elevated reactive oxygen species (ROS) generation, changes in the levels of antioxidant markers (MDA and GSH), and decreased expression of SLC7A11 and GPX4. The results of the present study clearly showed that PJME inhibited the proliferation of A549 cells and induced ferroptosis by reducing the expression of the important targets SLC7A11 and GPX4. Further research is necessary to fully understand the clinical efficacy of PJME before it can be investigated as supplemental or adjuvant therapy for lung cancer.

Noradrenaline Protects Human Microglial Cells (HMC3) Against Apoptosis and DNA Damage Induced by LPS and Aβ1-42 Aggregates In Vitro.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, characterized by the accumulation of amyloid-beta (Aβ) plaques and neuroinflammation. This study investigates the protective effects of noradrenaline (NA) on human microglial cells exposed to lipopolysaccharides (LPS) and Aβ aggregates-major contributors to inflammation and cellular damage in AD. The reduced Aβ aggregation in the HMC3 human microglial cells co-treated with Aβ and NA was confirmed by thioflavin T (ThT) assay, fluorescent ThT staining, and immunocytochemistry (ICC). The significantly increased viability of HMC3 cells after 48 h of incubation with NA at 50 µM, 25 µM, and 10 µM, exposed to IC50 LPS and IC50 Aβ, was confirmed by XTT and LDH assays. Moreover, we found that NA treatment at 25 μM and 50 μM concentrations in HMC3 cells exposed to IC50 LPS or IC50 Aβ results in an increased proliferation of HMC3 cells, their return to normal morphology, decreased levels of DNA damage, reduced caspase-3 activity, decreased expression of pro-apoptotic DDIT3 and BAX, and increased expression of anti-apoptotic BCL-2 genes and proteins, leading to enhanced cell survival, when compared to that of the HMC3 cells treated only with IC50 LPS or IC50 Aβ. Furthermore, we showed that NA induces the degradation of both extracellular and intracellular Aβ deposits and downregulates hypoxia-inducible factor 1α (HIF-1α), which is linked to impaired Aβ clearance and AD progression. These findings indicate that NA holds promise as a therapeutic target to address microglial dysfunction and potentially slow the progression of AD. Its neuroprotective effects, particularly in reducing inflammation and regulating microglial activity, warrant further investigation into its broader role in mitigating neuroinflammation and preserving microglial function in AD.

Pre-Clinical Study of Hydro-Ethanol (60:40) Extract of <i>Areca catechu</i> (L.) on Testicular Androgenesis in Albino Rat: An Approach for Herbal Male Contraceptive Development

The present study was designed to determine the anti-testicular activity of hydro-ethanol seed extract of Areca catechu (10, 20 and 40 mg) in dose-dependent fashion in albino rats. Seminal vesicular fructose (SVF), oxidative stress sensors, gene expression, somatic weight, reproductive organo-somatic indices, spermiological, hormonal profile, and testicular histological examination for qualitative and quantitative investigations of spermatogenesis were observed. In compared to the placebo treated control (PTC), the exposure of the said doses showed a statistical significant downward deviation in the spermiological, hormonal, SVF profile, kinetics of testicular key androgenic dehydrogenase enzymes, and diameters of seminiferous tubules. Anti-oxidant enzyme kinetics were reduced significantly against PTC, and the quantity of malondialdehyde in male gonad and sperm precipitate was elevated in significant level (p<0.05). Expression of apoptotic promoting gene in testis was increased (p<0.05) and anti-apoptotic Bcl-2 gene expression exhibited a significant decrease (p<0.05) in treated group comparison to the PTC. The alterations in the activities of phosphatases in hepatic tissue was not significant from the background of PTC suggesting that the plant has no systematic undesirable effect in physiological processes. The observation highlighted that 10 mg dose as the threshold dose for imposition of such anti- testicular activity leading to male contraception.

Investigation of apoptotic efficacy of propolis in MCF-7 cell line

Objective: Propolis, also known as bee glue, is a resinous compound collected by honey bees from various plants and processed by their saliva enzymes. Propolis and its components have been studied for their cytotoxic effects on cell lines in vitro, and recent studies have shown that they also have an antitumor effect in vivo. This study aimed to investigate the in-vitro apoptotic effects of propolis on the human breast cancer cell line (MCF-7). Method: The MTT test was used to determine the effect of propolis on cell viability and the doses to be administered. The GraphPad Prism Version 6.01 program was used to analyze the MTT results, while the qRT-PCR method was used to determine the expression levels of Caspase-8, Caspase-9, and Bcl-2 genes. The RT2 profiler PCR Assay Data Analysis version 3.5 was used to analyze gene expression data. Results: This study it was found that doses of 3.9 and 7.8 µg/ml of propolis showed no cytotoxic effect, while doses of 15.625 µg/ml and above had a cytotoxic effect. There was no change in the expression levels of genes at concentrations of 3.9 µg/ml and 7.8 µg/ml of propolis. However, at 15.625 µg/ml of propolis, Caspase-9 gene expression increased 11.89-fold (p=0.033). Although there was no significant difference in Caspase-8 gene expression in the extrinsic pathway of apoptosis (p=0.437), a 0.04-fold decrease in anti-apoptotic Bcl-2 gene expression was observed (p=0.000098). Conclusion: In conclusion, propolis showed a dose-dependent cytotoxic effect on the MCF-7 cell line, induced apoptosis, and did so via the intrinsic pathway of apoptosis. The study suggests that propolis has high potential as an anticancer agent since its apoptotic effects have been demonstrated in the MCF-7 cell line.

Extracellular vesicles from type-2 macrophages increase the survival of chronic lymphocytic leukemia cells ex vivo

The resistance of Chronic Lymphocytic Leukemia (CLL) B-cells to cell death is mainly attributed to interactions within their microenvironment, where they interact with various types of cells. Within this microenvironment, CLL-B-cells produce and bind cytokines, growth factors, and extracellular vesicles (EVs). In the present study, EVs purified from nurse-like cells and M2-polarized THP1 cell (M2-THP1) cultures were added to CLL-B-cells cultures. EVs were rapidly internalized by B-cells, leading to a decrease in apoptosis (P = 0.0162 and 0.0469, respectively) and an increased proliferation (P = 0.0335 and 0.0109). Additionally, they induced an increase in the resistance of CLL-B-cells to Ibrutinib, the Bruton kinase inhibitor in vitro (P = 0.0344). A transcriptomic analysis showed an increase in the expression of anti-apoptotic gene BCL-2 (P = 0.0286) but not MCL-1 and an increase in the expression of proliferation-inducing gene APRIL (P = 0.0286) following treatment with EVs. Meanwhile, an analysis of apoptotic protein markers revealed increased amounts of IGFBP-2 (P = 0.0338), CD40 (P = 0.0338), p53 (P = 0.0219) and BCL-2 (P = 0.0338). Finally, exploration of EVs protein content by mass spectrometry revealed they carry various proteins involved in known oncogenic pathways and the RNAseq analysis of CLL-B-cells treated or not with NLCs EVs show various differentially expressed genes.

The Effect of Prolactin on Gene Expression and the Secretion of Reproductive Hormones in Ewes during the Estrus Cycle.

Prolactin (PRL) plays an important role in animal follicle development and ovulation. However, its regulatory effects on the different stages of the estrus cycle in ewes are unclear. In this study, bromocriptine (BCR, PRL inhibitor) was used to study the effect of PRL on the secretion of reproductive hormones and gene expressions in order to explore its regulatory effects on the sexual cycle of ewes. Eighty healthy ewes with the same parity and similar weights were randomly assigned to the control group (C, n = 40) and the treatment group (T, n = 40, fed bromocriptine). After estrus synchronization, thirty-one ewes with overt signs of estrus were selected from each group. Six blood samples were randomly obtained by jugular venipuncture to measure the concentration of PRL, estrogen (E2), progesterone (P4), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and gonadotropin-releasing hormone (GnRH) in the proestrus, estrus, metestrus, and diestrus. At the same time, we collected the ovaries of the six ewes in vivo after anesthesia in order to detect follicle and corpus luteum (CL) counts and measure the expression of hormone-receptor and apoptosis-related genes. The results show that PRL inhibition had no significant effects on the length of the estrus cycle (p > 0.05). In proestrus, the number of large and small follicles, the levels of E2, FSH, and GnRH, and the expressions of ER, FSHR, and the apoptotic gene Caspase-3 were increased (p < 0.05); and the number of middle follicles and the expression of anti-apoptotic gene Bcl-2 were decreased (p < 0.05) in the T group. In estrus, the number of large follicles, the levels of E2 and GnRH, and the expressions of the StAR, CYP19A1, and Bcl-2 genes were increased (p < 0.05), and the number of middle follicles was decreased (p < 0.05) in the T group. In metestrus, the number of small follicles and the expression of LHR (p < 0.05) and the pro-apoptotic gene Bax were increased (p < 0.05); the number of middle follicles was decreased (p < 0.05) in the T group. In diestrus, the number of large follicles, middle follicles, and CL, the level of P4, and the expressions of PR, 3β-HSD, StAR, Caspase-3, and Bax were increased (p < 0.05); the number of small follicles and the expression of Bcl-2 were decreased (p < 0.05) in the T group. In summary, PRL inhibition can affect the secretion of reproductive hormones, the follicle count, and the gene expression during the estrus cycle. These results provide a basis for understanding the mechanisms underlying the regulation of the ewe estrus cycle by PRL.

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 &lt; 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

Kidney-tonifying formula facilitates the development and maturation of mouse preantral follicle in vitro.

Kidney-tonifying formulas are frequently used in clinical practices to enhance follicular development and maturation. This research explored the impacts of the Bushen Tiaojing formula (BSTJF) on the development of mouse preantral follicles in vitro and its relationship with granulosa cells and gonadotropins. Preantral follicles were extracted from mice and cultured with or without serum from rats that were previously treated with or without BSTJF. During cultivation, the follicles were monitored for morphological changes and developmental maturation. Exhausted medium was collected every other day for the measurement of progesterone and estradiol (E2) levels by ELISA. Granulosa cells in in-vitro medium were collected on days 8, 10, and 12 and analyzed for determining the expressions of apoptosis-associated genes (Bax, Bcl-2, and Caspase-3). Propagation and apoptosis rates of collected granulosa cells were measured by CCK-8 assay and flow cytometry. Compared with control follicles, follicles cultured with serum from BSTJF-treated rats had a higher survival rate, larger follicle diameter, higher Bcl-2 expression, and lower Bax and Caspase-3 expressions (all P ≤ 0.05). In addition, their granulosa cells presented substantially elevated proliferation (P ≤ 0.05) and a lower rate of apoptosis (P ≤ 0.05) compared with granulosa cells from control follicles. The level of E2 in the culture media of all groups increased slowly in the first 6 days. Subsequently, after formation of the antrum, the levels of E2 and progesterone were enhanced in the medium of follicles cultured with serum from BSTJF-treated rats compared with those in the media of control follicles (all P ≤ 0.05). Serum from BSTJF-treated rats facilitated the in vitro development and maturation of mouse follicles by increasing the expression of anti-apoptotic gene Bcl-2, reducing the expressions of pro-apoptotic genes Bax and Caspase-3 as well as the apoptosis of granulosa cells, promoting the proliferation of granulosa cells and increasing the secretion of E2 and progesterone in the cells.

Evaluation of the Cytotoxicity of Secondary Bioactive Compounds Produced by Streptomyces in Soil against a Colon Cancer Cell Line.

Colorectal cancer is one of the most serious malignancies affecting humans. In this study, Streptomyces bioactivechemicals extracted from soil were analyzed for their anti-colorectal-cancer and antibacterial properties. A total of 100 soil samples were collected from Kerman-Iran, incubated in SCA media and the antimicrobial properties were tested using the cross-streak method. Three strains were cultured in ISP4 medium to obtain secondary bioactive compounds. After studying the effects of the bioactive compounds on the HT29 and human foreskin fibroblast (HFF) cell lines, the expression of the p53, p21, BAX, BCL2, Casp3 and Casp8 genes was analyzed by real-time PCR and flow cytometry to detect the presence of apoptosis.The isolates show high degree of identification with Streptomyces rochei, Streptomyces fungicidicus and Streptomyces maritimus due to 16SrDNA sequence homology. Compared to HT-29 cells, Streptomyces extracts had lower cytotoxicity against normal cells (SI=5.88), followed by HFF (SI=4.14). The cell lines demonstrated a dose-dependent significant increase in DNA fragmentation, an increase in the proportion of cells in sub-G1 phase and caused G2/M cell cycle arrest in HT-29 and HFF cells.The bacterial extracts obtained displayed strong antibacterial properties and inhibited the proliferation of HT-29 and HFF cell lines. The treated cells exhibited morphological changes caused by the activation of caspase and p53/p21 proteins. This confirms that Streptomyces-induced apoptosis is mediated by the activation of p21/p53. Anti-apoptotic Bcl-2 gene expression was downregulated by treatment with the extracts. Further studies are needed to understand the antimicrobial properties of Streptomyces.

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.