Bcl-2 Activation Research Articles

Upregulation of BAX and caspase-3, as well as downregulation of Bcl-2 during treatment with indeno[1,2-b]quinoxalin derivatives, mediated apoptosis in human cancer cells.

Cancer is the world's foremost cause of death. There are over 100 different forms of cancer. Cancers are frequently named after the organs or tissues in which they develop. As a part of our aim to develop promising anticancer agents, a series of new indeno[1,2-b]quinoxaline derivatives were synthesized. All of the synthesized compounds were tested for anticancer activity in vitro in three human cancer cell lines: the HCT-116 colon cancer cell line, the HepG-2 liver cancer cell line, and the MCF-7 breast cancer cell line. Among the tested derivatives, 2, 3, 5, 12, 21, and 22 showed exceptional antiproliferative activities against the three tested cell lines compared to the reference standard imatinib. These compounds were, therefore, selected for further investigations. Evaluation of their cytotoxicity against a normal human cell line (WI-38) was performed, to ensure their safety and selectivity (IC50 > 92 μM). Then, induction of apoptosis by the most active compounds was found to be accomplished by downregulation of Bcl-2 and upregulation of BAX and caspase-3. After that, the most promising apoptotic compound that increases the caspase-3 and BAX expression and downregulates Bcl-2 activity (3) was assessed for its impact on the cell cycle distribution in HepG-2 cells: The most potent derivative (3) induced cell cycle arrest at the G2/M phase. Finally, in silico evaluation of the ADME properties indicated that compound 3 is orally bioavailable and can be readily synthesized on a large scale.

Lycium barbarum polysaccharide attenuates Pseudomonas-aeruginosa pyocyanin-induced cellular injury in mice airway epithelial cells.

BackgroundLycium barbarum berries have been utilized in Asia for many years. However, the mechanisms of its lung-defensive properties are indeterminate.ObjectiveWe investigate whether L. barbarum polysaccharide (LBP) could weaken Pseudomonas aeruginosa infection-induced lung injury.DesignMice primary air-liquid interface epithelial cultures were pretreated with LBP and subsequently treated with pyocyanin (PCN). Lung injury, including apoptosis, inflammation, and oxidative stress, was estimated by western blot, enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction, Real-time qPCR (Q-PCR). Flow cytometry was used to test cell apoptosis. Moreover, Balb/c mice were used to evaluate the tissue injury. We used hematoxylin-eosin staining and immunofluorescence to detect the expression of related proteins and tissue damage in mouse lungs and spleen.ResultsThe flow cytometric analysis shows the potential of LBP to reduce time-dependent cell death by PCN. Mechanistically, LBP reduces PCN-induced expression of proapoptotic proteins and caspase3 and induces the activation of Bcl-2 in mice bronchial epithelial cells. Similarly, LBP reduces PCN-induced intracellular reactive oxygen species (ROS) production. Moreover, LBP inhibits the production of inflammatory cytokines, Interleukin (IL-1β), Tumor Necrosis Factor (TNF), IL-6, and IL-8. Our study confirms the ability of LBP to retard PCN-induced injury in mice lung and spleen.ConclusionsThe inhibition of PCN-induced lung injury by LBP is capable of protecting mice cells from injury.

Effects of swimming training on myocardial protection in rats.

Swimming is important for promoting and maintaining health, as it can increase the efficiency of the cardiovascular system and decrease the occurrence of cardiovascular diseases. The objective of the present study was to examine whether swimming training could decrease myocardial injury in rats caused by myocardial ischemia/reperfusion (I/R). Sprague-Dawley rats were randomized into four groups, namely the Sham, coronary artery occlusion, swimming training and ischemic preconditioning (IPC) groups. Myocardial I/R was induced in anesthetized male Sprague-Dawley rats by a 40-min occlusion followed by a 3-h reperfusion of the left anterior descending coronary artery. The rats were sacrificed after surgery and their hearts were examined. The results demonstrated that the number of TUNEL-positive nuclei and degree of caspase-3 activation were both significantly increased in the myocardium following myocardial I/R in rats, indicating increased cardiomyocyte apoptosis. On the other hand, swimming training decreased the serum levels of creatine phosphokinase, lactate dehydrogenase and cardiac troponin I, and was associated with reduced histological damage and myocardial infarct size. Furthermore, swimming training also reduced TNF-α levels, caspase-3 activation and enhanced Bcl-2 activation, which decreased the number of apoptotic cells in the myocardium. The findings of the present study showed that swimming training and IPC could similarly decrease myocardial injury following myocardial I/R, and may therefore be used as exercise training to effectively prevent myocardial injury.

CO2 pneumoperitoneum effects on proliferation and apoptosis in two different neuroblastoma cell lines.

The proto-oncogene MYCN is considered a transcription factor involved in the regulation of neuroblastoma (NB) cell biology. Since minimally invasive-surgery represents a debated treatment of NB, we investigated CO2 effects on proliferative activity and apoptotic pathway in two NB cell lines, SH-SY5Y (MYCN-non-amplified) and IMR-32 (MYCN-amplified). SH-SY5Y and IMR-32 were exposed to CO2 (100%) at a pressure of 15mmHg for 4h and then moved to normal condition for 24h. Cell proliferation, caspase 3 activity and transcript levels of BAX, BCL-2, cyclin B, cyclin D and MMP-2 were evaluated. CO2 exposure caused a decrease in cell proliferation associated to increases in BAX/BCL-2 ratio and caspase 3 activity in SH-SY5Y, while opposite effects have been found in IMR-32. CO2 exposure induced a decrease of cyclin B1 in SH-SY5Y, while an increase in cyclin B1 and D1 was observed in IMR-32. A slight up-regulation of MMP-2 expression in SH-SY5Y and a significant increase of 2.2 folds in IMR-32 was observed (p < 0.05). Our results suggest that CO2 exposure may cause different effects on various NB cell lines, likely due toMYCN amplification status. Further in vitro and in vivo studies are needed to highlight the role of laparoscopy on NB behaviour.

Study on the protective effect and mechanism of Liriodendrin on radiation enteritis in mice.

Patients receiving pelvic or abdominal radiotherapy may experience acute and/or chronic side effects due to gastrointestinal changes. However, effective medicine for treating radiation enteritis has not been found yet. Sargentodoxa cuneata is a famous Chinese medicine used to treat intestinal inflammation, and our research team has found the main biologically active compound through its extraction, which is Liriodendrin. In this study, we found that Liriodendrin can reduce the expression of Cer, Cer1P and S1P in the sphingolipid pathway, thereby reducing the histological damage to the intestinal tract of mice and inhibiting the apoptosis of intestinal tissue cells. In addition, Liriodendrin can reduce the levels of pro-inflammatory cytokines (IL-6 and TNF-α), and it is suggested through flow cytometry that the proportion of neutrophils in the intestinal tissue can decrease due to the existence of Liriodendrin. At the same time, the western blot evaluation revealed that Liriodendrin significantly inhibited the activation of Bcl-2/Bax/Caspase-3 and NF-κB signaling pathways. The results show that Liriodendrin can inhibit intestinal inflammation and intestinal cell apoptosis through the sphingolipid pathway. Therefore, the aforementioned results demonstrated that Liriodendrin may be a promising drug for the treatment of radiation enteritis.

Cytotoxic Activity, and Molecular Docking of Indole Alkaloid Voacangine and Bisindole Alkaloid Vobtusine, Vobtusine Lactone from the Indonesian Plant: Voacanga foetida (Blume) Rolfe

ABSTRACT&#x0D; The purpose of this study was to isolate and test its cytotoxic activity starting from extract fraction and its isolate compound, then carried out molecular docking to confirm the potential biological activity of ligands (vocangine, vobtusine, and vobtusine lactone) against inhibition proteins (Bcl-2, Bcl-xL, and Mcl-1), activation protein Bax and activation of apoptotic execution protein Caspase-3. This research is an experimental quantitative study using column chromatography and HPLC methods in the isolation process, MTT assay in determining the cytotoxic activity, and molecular docking in determining the prediction of apoptotic mechanism. The cytotoxic activity of VFB-DA, VFB-DB, VFB-BuOH, VFB-DB4 fractions, voacangine compounds, vobtusine are very strong. while vobtusine lactone is moderate cytotoxic activity. The docking score for voacangine, vobtusine, and vobtusine lactone compounds against Bcl-2 is -9.93; -10.07; -9.03 kcal/mol, against Bcl-xl is -9.77; -11.69; -9.76 kcal/mol, against Mcl-1 is -10.70; -10.77; -9.53 kcal/mol, and for Bax is -8.99; -6.87; -6.99 kcal/mol, as well as against caspase3 is -12.05; -12.21; -12.02 kcal/mol. The cytotoxic activity of voacangine, vobtusine, and vobtusine lactone compounds is thought to cause cell death by suppressing Bcl-2 activity; Bcl-xl; and Mcl-1, increased Bax activity and increased caspase3 activity.&#x0D; &#x0D; Key words: Voacanga foetida, in vitro, in silico

Cytotoxic Effect of Hydroethanolic Extract of Citrullus colocynth (L) Fruit against Human Lung Cancer Cell Line

Citrullus colocynthis is a traditional medicinal plant that belongs to the cucurbitaceae family. The extract of the fruit is rich in therapeutic phytochemicals. Lung cancer is a disease with high mortality and morbidity rates. Recently the research has been focused on herbal medicine as a treatment approach for cancer because of its no or less side effects. The aim of the study is to know about the anticancer effect of hydroethanolic extract of Citrullus colocynthis fruit against human lung cancer cell line through apoptosis pathway. Cell viability test was done by MTT assay. The mRNA expression of Bcl2 and Bcl-xL was done using the real-time PCR. The obtained data was analysed statistically by one way analysis of variance and Duncan multiple range test with graph prism version 5 to analyse the significance. The significance was considered at p&lt;0.05 level in Duncan’s test. Citrullus colocynthis fruit extract increased the inhibition of growth of lung cancer cells. The activity of Bcl2 and Bcl xL was significantly down regulated at 400μg. The study concludes that Citrullus colocynthis fruit extract has anticancer activity on A549 human lung cancer cell line through apoptosis pathway.

Targeting STAT3 prevents bile reflux-induced oncogenic molecular events linked to hypopharyngeal carcinogenesis.

The signal transducer and activator of transcription 3 (STAT3) oncogene is a transcription factor with a central role in head and neck cancer. Hypopharyngeal cells (HCs) exposed to acidic bile present aberrant activation of STAT3, possibly contributing to its oncogenic effect. We hypothesized that STAT3 contributes substantially to the bile reflux‐induced molecular oncogenic profile, which can be suppressed by STAT3 silencing or pharmacological inhibition. To explore our hypothesis, we targeted the STAT3 pathway, by knocking down STAT3 (STAT3 siRNA), and inhibiting STAT3 phosphorylation (Nifuroxazide) or dimerization (SI3‐201; STA‐21), in acidic bile (pH 4.0)‐exposed human HCs. Immunofluorescence, luciferase assay, Western blot, enzyme‐linked immunosorbent assay and qPCR analyses revealed that STAT3 knockdown or pharmacologic inhibition significantly suppressed acidic bile‐induced STAT3 activation and its transcriptional activity, Bcl‐2 overexpression, transcriptional activation of IL6, TNF‐α, BCL2, EGFR, STAT3, RELA(p65), REL and WNT5A, and cell survival. Our novel findings document the important role of STAT3 in bile reflux‐related molecular oncogenic events, which can be dramatically prevented by STAT3 silencing. STA‐21, SI3‐201 or Nifuroxazide effectively inhibited STAT3 and cancer‐related inflammatory phenotype, encouraging their single or combined application in preventive or therapeutic strategies of bile reflux‐related hypopharyngeal carcinogenesis.

Combined Inhibition of Bcl2 and Bcr-Abl1 Exercises Anti-Leukemia Activity but Does Not Eradicate the Primitive Leukemic Cells.

Background: The management of Philadelphia Chromosome-positive (Ph+) hematological malignancies is strictly correlated to the use of BCR-ABL1 tyrosine kinase inhibitors (TKIs). However, these drugs do not induce leukemic stem cells death and their persistence may generate a disease relapse. Published reports indicated that Venetoclax, a selective BCL2 inhibitor, could be effective in Ph+ diseases, as BCL2 anti-apoptotic activity is modulated by BCR-ABL1 kinase. We, therefore, investigated if BCL2 inhibition, alone or combined with Nilotinib, a BCR-ABL1 inhibitor, affects the primitive and committed Ph+ cells survival. Methods: We used Ph+ cells isolated from leukemic patients at diagnosis. To estimate the therapeutic efficacy of BCL2 and BCR-ABL1 inhibition we employed long-term culture, proliferation and apoptosis assay. Immunoblot was used to evaluate the ability of treatment to interfere with the down-stream targets of BCR-ABL1. Results: Blocking BCL2, we observed reduced proliferation and clonogenic potential of CML CD34-positive cells and this cytotoxicity was improved by combination with BCR-ABL1 inhibitor. However, BCL2 inhibition, alone or in combination regiment with BCR-ABL1 inhibitor, did not reduce the self-renewal of primitive leukemic cells, while strongly induced cell death on primary Ph+ Acute Lymphoblastic Leukemia (ALL). Conclusion: Our results suggest that primitive CML leukemic cells are not dependent on BCL2 for their persistence and support that committed CML and Ph + ALL cells are dependent by BCL2 and BCR-ABL1 cooperation for their survival. The antileukemic activity of BCL2 and BCR-ABL1 dual targeting may be a useful therapeutic strategy for Ph+ ALL patients.

Targeting mitochondrial respiration and the BCL2 family in high-grade MYC-associated B-cell lymphoma.

Multiple molecular features, such as activation of specific oncogenes (e.g., MYC, BCL2) or a variety of gene expression signatures, have been associated with disease course in diffuse large B‐cell lymphoma (DLBCL), although their relationships and implications for targeted therapy remain to be fully unraveled. We report that MYC activity is closely correlated with—and most likely a driver of—gene signatures related to oxidative phosphorylation (OxPhos) in DLBCL, pointing to OxPhos enzymes, in particular mitochondrial electron transport chain (ETC) complexes, as possible therapeutic targets in high‐grade MYC‐associated lymphomas. In our experiments, indeed, MYC sensitized B cells to the ETC complex I inhibitor IACS‐010759. Mechanistically, IACS‐010759 triggered the integrated stress response (ISR) pathway, driven by the transcription factors ATF4 and CHOP, which engaged the intrinsic apoptosis pathway and lowered the apoptotic threshold in MYC‐overexpressing cells. In line with these findings, the BCL2‐inhibitory compound venetoclax synergized with IACS‐010759 against double‐hit lymphoma (DHL), a high‐grade malignancy with concurrent activation of MYC and BCL2. In BCL2‐negative lymphoma cells, instead, killing by IACS‐010759 was potentiated by the Mcl‐1 inhibitor S63845. Thus, combining an OxPhos inhibitor with select BH3‐mimetic drugs provides a novel therapeutic principle against aggressive, MYC‐associated DLBCL variants.

Co-Targeting Intrinsic and Extrinsic Apoptosis to Maximize Cell Death Induction in Venetoclax-Resistant AML Cells

Bcl-2 family protein-regulated intrinsic and IAP-family protein-regulated extrinsic pathways are two major apoptotic cell death mechanisms. Components of both pathways are regulated by the tumor suppressor p53. Although combinations of Bcl-2 inhibitor venetoclax (VEN) and a hypomethylating agent induce high response rates in AML, most patients ultimately relapse. In addition, pre-clinical and clinical studies have shown that TP53-mutant AML cells are less sensitive to VEN (Carter BZ, ASH 2019 and 2020; DiNardo CD, Blood 2020). We investigated if simultaneous inhibition of Bcl-2 and IAPs and activation of p53, via MDM2 inhibition could maximize apoptosis induction in AML cells with acquired resistance to VEN-based therapy or in those carrying TP53 mutations.We treated MV4-11 cells with acquired resistance to VEN (VEN-R) with the Bcl-2 inhibitor APG2575, IAP inhibitor APG1387, or MDM2 inhibitor APG115, and with their combinations. As expected, VEN-R cells were more resistant to APG2575 compared to control cells. APG1387 alone had limited activity in both VEN-R and in control cells and the combination of APG2575 and APG1387 enhanced cell killing (P < 0.05 compared to each single agent). APG115 was active in both VEN-R and control cells and its activity was only increased by APG2575 in the control cells, but minimally affected by either APG2575 or APG1387 in VEN-R cells. However, maximal apoptosis induction was observed in both VEN-R and control cells when all three compounds were combined (P < 0.05 compared to any of the double combinations or single agent treatments).We next treated NSG mice harboring PDX cells derived from an AML patient who relapsed on the VEN/decitabine therapy with APG2575 (50 mg/kg, p.o., daily), APG1387 (10 mg/kg, i.v., once/wk), APG115 (50 mg/kg, p.o., daily at wk 1 and 5), or combinations. At the end of a 5-wk treatment, significant reductions of human CD45 + cells were observed in all treatment groups. At 4 wks post treatment, decreased circulating leukemia cells were found in the triple and APG115+APG2575 combination groups. APG115 (139 d, p=0.009), APG1387 (130 d, p=0.004), or APG2575 (132 d, p=0.004) significantly extended mouse survival compared to controls (116 d). Among two drug combinations, APG115 plus APG1387 did not further prolong survival, APG2575 plus APG1387 (144 d, p<0.01) was more effective, and APG2575 plus APG115 (180 d, p<0.01) most effectively extended survival compared to each drug alone. Triple combination treated mice lived longest (185 d), which was significantly longer than APG115 plus APG1387 and APG2575 plus APG1387 but did not reach statistical significance compared to APG2575 plus APG115. Data showed that triple and APG115+APG2575 combinations were most effective, followed by APG2575+APG1387, then APG115+APG1387, APG2575, or APG115, and finally APG1387.Finally, we treated Molm13 cells lacking TP53 or carrying TP53 mutations (R248W/R213*, R248Q, R175H, R282W, Y220C) with the three agents and their combinations. All mutant cells were insensitive to single drugs. Enhanced activity was observed when any of two agents were combined and combined inhibition of Bcl-2, IAPs, and MDM2 most effectively induced cell death in TP53 knockout and all TP53 mutant cells (P < 0.05 for the triple combination compared to any of the double combinations or single agent treatments, and double combinations compared to their respective single agent treatments). Western blot analysis showed that decreased cIAP1, cIAP2, XIAP, or p21 was observed in single agent or combination-treated cells. Only in the triple combination group, cIAP1, cIAP2, and XIAP as well as MDM2 were largely diminished and p21 was marked decreased.In conclusion, our study demonstrates that co-targeting intrinsic and extrinsic apoptosis maximizes cell death induction in AML cells with acquired resistance to VEN or with TP53 deletion/mutations by antagonizing Bcl-2, eliminating cIAPs and XIAP, as well as MDM2 and p21, a finding that needs to be validated clinically. DisclosuresCarter: Syndax: Research Funding; Ascentage: Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Andreeff: Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Medicxi: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees.

Total flavonoids of Drynariae Rhizoma regulates ER-p38 MAPK signaling pathway to improve scopolamine-induced learning and memory impairments in model mice

This study intended to explore the effect and mechanism of total flavonoids of Drynariae Rhizoma in improving scopola-mine-induced learning and memory impairments in model mice. Ninety four-month-old Kunming(KM) mice were randomly divided into six groups. The ones in the model group and blank group were treated with intragastric administration of normal saline, while those in the medication groups separately received the total flavonoids of Drynariae Rhizoma, Kangnaoshuai Capsules, donepezil, as well as total flavonoids of Rhizoma Drynariae plus estrogen receptor(ER) blocker by gavage. The mouse model of learning and memory impairments was established via intraperitoneal injection of scopolamine. Following the measurement of mouse learning and memory abilities in Morris water maze test, the hippocampal ERβ expression was detected by immunohistochemistry, and the expression levels of ERβ and phosphorylated p38(p-p38) in the hippocampus and B-cell lymphoma 2(Bcl-2), Bcl-2-associated death promoter(Bad), and cysteinyl aspartate-specific protease-3(caspase-3) in the apoptotic system were assayed by Western blot. The contents of malondia-ldehyde(MDA), superoxide dismutase(SOD), and nitric oxide(NO) in the hippocampus were then determined using corresponding kits. Compared with the control group, the model group exhibited significantly prolonged incubation period, reduced frequency of cros-sing the platform, shortened residence time in the target quadrant, lowered ERβ, Bcl-2 and SOD activity in the hippocampus, and increased p-p38/p38, Bad, caspase-3, MDA, and NO. Compared with the model group, the total flavonoids of Rhizoma Drynariae increased the expression of ERβ and SOD in the hippocampus, down-regulated the expression of neuronal pro-apoptotic proteins, up-re-gulated the expression of anti-apoptotic proteins, and reduced p-p38/p38, MDA, and NO. The effects of total flavonoids of Drynariae Rhizoma on the above indexes were reversed by ER blocker. It has been proved that the total flavonoids of Drynariae Rhizoma obviously alleviate scopolamine-induced learning and memory impairments in mice, which may be achieved by regulating the neuronal apoptotic system and oxidative stress via the ER-p38 mitogen-activated protein kinase(ER-p38 MAPK) signaling pathway.

Anti-Apoptotic Effect of Flavokawain A on Ochratoxin-A-Induced Endothelial Cell Injury by Attenuation of Oxidative Stress via PI3K/AKT-Mediated Nrf2 Signaling Cascade.

This study investigates the endothelial protective activity of flavokawain A (FKA) against oxidative stress induced by ochratoxin A (OTA), which acts as a mycotoxin, and its primary mechanisms in in vitro models. Reactive oxygen species, in general, regulate oxidative stress that significantly contributes to the pathophysiology of endothelial dysfunctions. OTA exerts toxicity through inflammation and the accumulation of ROS. This research is aimed at exploring the defensive function of FKA against the endothelial injury triggered by OTA through the Nrf2 pathway regulated by PI3K/AKT. OTA exposure significantly increased the nuclear translocation of NFκB, whereas we found a reduction in inflammation via NFκB inhibition with FKA treatment. FKA increased the PI3K and AKT phosphorylation, which may lead to the stimulation of antioxidative and antiapoptotic signaling in HUVECs. It also upregulated the phosphorylation of Nrf2 and a concomitant expression of antioxidant genes, such as HO-1, NQO-1, and γGCLC, depending on the dose under the oxidative stress triggered by OTA. Knockdown of Nrf2 through small interfering RNA (siRNA) impedes the protective role of FKA against the endothelial toxicity induced by OTA. In addition, FKA enhanced Bcl2 activation while suppressing apoptosis marker proteins. Therefore, FKA is regarded as a potential agent against endothelial oxidative stress caused by the deterioration of the endothelium. The research findings showed that FKA plays a key role in activating the p-PI3K/p-AKT and Nrf2 signaling pathways, while suppressing caspase-dependent apoptosis.

SNHG8 Promotes the Progression of Epstein-Barr Virus-Associated Gastric Cancer via Sponging miR-512-5p and Targeting TRIM28.

SNHG8, a family member of small nucleolar RNA host genes (SNHG), has been reported to act as an oncogene in gastric carcinoma (GC). However, its biological function in Epstein–Barr virus (EBV)-associated gastric cancer (EBVaGC) remains unclear. This study investigated the role of SNHG8 in EBVaGC. Sixty-one cases of EBVaGC, 20 cases of non-EBV-infected gastric cancer (EBVnGC), and relative cell lines were studied for the expression of SNHG8 and BHRF1 (BCL2 homolog reading frame 1) encoded by EBV with Western blot and qRT-PCR assays. The relationship between the expression levels of SNHG8 and the clinical outcome in 61 EBVaGC cases was analyzed. Effects of overexpression or knockdown of BHRF1, SNHG8, or TRIM28 on cell proliferation, migration, invasion, and cell cycle and the related molecules were determined by several assays, including cell proliferation, colony assay, wound healing assay, transwell invasion assay, cell circle with flow cytometry, qRT-PCR, and Western blot for expression levels. The interactions among SNHG8, miR-512-5p, and TRIM28 were determined with Luciferase reporter assay, RNA immunoprecipitation (RIP), pull-down assays, and Western blot assay. The in vivo activity of SNHG8 was assessed with SNHG8 knockdown tumor xenografts in zebrafish. Results demonstrated that the following. (1) BHRF1 and SNHG8 were overexpressed in EBV-encoded RNA 1-positive EBVaGC tissues and cell lines. BHRF1 upregulated the expressions of SNHG8 and TRIM28 in AGS. (2) SNHG8 overexpression had a significant correlation with tumor size and vascular tumor thrombus. Patients with high SNHG8 expression had poorer overall survival (OS) compared to those with low SNHG8 expression. (3) SNHG8 overexpression promoted EBVaGC cell proliferation, migration, and invasion in vitro and in vivo, cell cycle arrested at the G2/M phase via the activation of BCL-2, CCND1, PCNA, PARP1, CDH1, CDH2 VIM, and Snail. (4) Results of dual-luciferase reporter assay, RNA immunoprecipitation, and pull-down assays indicated that SNHG8 sponged miR-512-5p, which targeted on TRIM28 and promoted cancer malignant behaviors of EBVaGC cells. Our data suggest that BHRF1 triggered the expression of SNHG8, which sponged miR-512-5p and upregulated TRIM28 and a set of effectors (such as BCL-2, CCND1, CDH1, CDH2 Snail, and VIM) to promote EBVaGC tumorigenesis and invasion. SNHG8 could be an independent prognostic factor for EBVaGC and sever as target for EBVaGC therapy.

Knockdown of LKB1 Sensitizes Endometrial Cancer Cells via AMPK Activation.

Metformin is an anti-diabetic drug and has anticancer effects on various cancers. Several studies have suggested that metformin reduces cell proliferation and stimulates cell-cycle arrest and apoptosis. However, the definitive molecular mechanism of metformin in the pathophysiological signaling in endometrial tumorigenesis and metastasis is not clearly understood. In this study, we examined the effects of metformin on the cell viability and apoptosis of human cervical HeLa and endometrial HEC-1-A and KLE cancer cells. Metformin suppressed cell growth in a dose-dependent manner and dramatically evoked apoptosis in HeLa cervical cancer cells, while apoptotic cell death and growth inhibition were not observed in endometrial (HEC-1-A, KLE) cell lines. Accordingly, the p27 and p21 promoter activities were enhanced while Bcl-2 and IL-6 activities were significantly reduced by metformin treatment. Metformin diminished the phosphorylation of mTOR, p70S6K and 4E-BP1 by accelerating adenosine monophosphate-activated kinase (AMPK) in HeLa cancer cells, but it did not affect other cell lines. To determine why the anti-proliferative effects are observed only in HeLa cells, we examined the expression level of liver kinase B1 (LKB1) since metformin and LKB1 share the same signalling system, and we found that the LKB1 gene is not expressed only in HeLa cancer cells. Consistently, the overexpression of LKB1 in HeLa cancer cells prevented metformin-triggered apoptosis while LKB1 knockdown significantly increased apoptosis in HEC-1-A and KLE cancer cells. Taken together, these findings indicate an underlying biological/physiological molecular function specifically for metformin-triggered apoptosis dependent on the presence of the LKB1 gene in tumorigenesis.

Effects of taxifolin on bone formation and apoptosis in experimental periodontitis in diabetic rats

ABSTRACT We investigated the therapeutic potential of taxifolin for treatment of alveolar bone loss (ABL) in experimental periodontitis in diabetic rats. Diabetes mellitus (DM) was induced by streptozotocin. Rats were divided into six groups: untreated control; DM only (D) group; ligature only (P) group; DM + ligature (DP) group; DM + ligature + 5 mg/kg/day taxifolin (Taxi-5) group; DM + ligature + 10 mg/kg/day taxifolin (Taxi-10) group. Experimental periodontitis was induced by ligation of the first molar and allowed to progress for 30 days before performing cone-beam computed tomographic (CBCT), histomorphometric and immunohistochemical analyses of periodontal tissue destruction. ABL was assessed using CBCT. ABL was greatest in the P and DP groups. Decreased ABL was observed in the Taxi-5 and Taxi-10 groups. Bone morphogenic protein (BMP-2), osteocalcin (OCN), receptor activator of nuclear factor kappa-Β ligand (RANKL), alkaline phosphatase (ALP), type I collagen, B cell lymphoma-associated X (Bax), and B-cell lymphoma 2 (Bcl-2) levels were investigated using immunohistochemistry. The Taxi-5 and Taxi-10 groups exhibited decreased RANKL expression, but increased BMP-2, ALP, type I collagen and OCN levels compared to the P and DP groups. Bax activity was increased in the D, P and DP groups. Taxi-5 and Taxi-10 groups exhibited increased Bcl-2 activity. Our findings suggest that taxifolin can reduce apoptosis and improve alveolar bone formation in diabetic rats with periodontitis.

Metformin and Sildenafil Attenuate Inflammation and Suppress Apoptosis After Ischemia/Reperfusion Injuries in Rat Urinary Bladder.

PurposeAlthough metformin and sildenafil can protect various organs against ischemia/reperfusion (I/R) injuries, their effects and mechanisms of action in bladder I/R injuries remain unknown. This study investigated the effects and mechanisms of action of metformin and sildenafil against bladder I/R insults in rats.MethodsOne hundred male Sprague-Dawley rats were randomly divided into 5 groups, each of which contained 20 rats: a sham-operated group, a bladder I/R group, and bladder I/R groups treated with metformin, sildenafil, or both agents. Ischemia was induced by clamping the bilateral common iliac arteries with atraumatic vascular clamps for 2 hours, followed by reperfusion for 7 days. During this period, rats were injected once daily with 4-mg/kg metformin and/or 1-mg/kg sildenafil.ResultsI/R injuries induced increased malondialdehyde levels and myeloperoxidase activity and decreased superoxide dismutase activity. These changes were attenuated by treatment with metformin and/or sildenafil. The I/R group had significantly higher Jun N-terminal kinase, p38 mitogen-activated protein kinase (MAPK), Bax, caspase-3, and nuclear factor-kappa B (NF-κB) levels, and lower extracellular signal-regulated kinase, and Bcl-2 levels in the bladder than the sham-operated group; these changes were significantly ameliorated by metformin and/or sildenafil treatment. No differences in the levels of these markers were observed between rats coadministered metformin and sildenafil and those treated with either agent alone.ConclusionsMetformin and sildenafil protected the rat bladder against I/R injuries. This effect may have been due to the inhibition of reactive oxygen species production through MAPK, Bax, and Bcl-2 activation, and the restoration of inflammation through NF-κB inhibition. However, the combination of metformin and sildenafil was not more effective than either agent alone.

Inflammatory markers in chronic kidney disease and end stage renal disease patients.

Chronic kidney disease (CKD) is condition characterized by a gradual loss of kidney function, patient with CKD suffering from a variety of immune system defects. This study looked at Fas, T cell, BCl2, and P53 activity in people with CKD, end stage renal disease (ESRD), and stable controls. The CD4+ and CD8+ levels in ESRD patients' peripheral blood were slightly lower than those in CKD patients. The CKD and ESRD groups had slightly higher Fas and FasL mRNA expression and slightly lower BCl2 mRNA gene expression than the normal control group (P < 0.05). P53 mRNA gene expression was shown to be higher in the patients than in the controls (P < 0.01). ESRD patients have a significantly lower number of T-cell subsets than CKD patients this is related to a higher degree of apoptosis in these cells.

Evaluation of Synthetic 2,4-Disubstituted-benzo[g]quinoxaline Derivatives as Potential Anticancer Agents.

A new series of 2,4-disubstituted benzo[g]quinoxaline molecules have been synthesized, using naphthalene-2,3-diamine and 1,4-dibromonaphthalene-2,3-diamine as the key starting materials. The structures of the new compounds were confirmed by spectral data along with elemental microanalyses. The cytotoxic activity of all synthesized benzo[g]quinoxaline derivatives was assessed in vitro against the breast MCF-7 cancer cell line. The tested molecules revealed good cytotoxicity toward the breast MCF-7 cancer cell line, especially compound 3. The results of topoisomerase IIβ inhibition assay revealed that compound 3 exhibits potent inhibitory activity in submicromolar concentration. Additionally, compound 3 was found to cause pre-G1 apoptosis, and slightly increase the cell population at G1 and S phases of the cell cycle profile in MCF-7 cells. Finally, compound 3 induces apoptosis via Bax activation and downregulation of Bcl2, as revealed by ELISA assay.

Flow Cytometry Detection of Sperm DNA Fragmentation and Apoptotic Markers in the Semen of Infertile Males.

The effect of sperm molecular defects on fertilization and pregnancy outcome after assisted reproductive therapy (ART) is widely documented by both research and clinical societies. Sperm DNA fragmentation and abnormal chromatin condensation represent critical causes of male infertility. Advanced androgenic techniques for accurately identifying molecular defects help in selecting an appropriate treatment strategy. Additionally, specific markers of apoptosis are increasingly important in predicting male infertility. The ability of flow cytometry to estimate the quantity of sperm with DNA fragmentation or damage and multifactor measurements in immotile sperm have made this developed technique essential in fertility centers. The study is aimed at assessing the level of DNA fragmentation and apoptosis by measuring flow cytometry using new techniques. Flow cytometry analysis revealed a varying degree of DNA damage. It was able to quantify the degree of impairment even in samples with minimal DNA fragmentation. DNA damage was observed even in samples that were considered normal with a routine semen analysis. Flow cytometry was sensitive to changes in sperm apoptosis. Elevated p53 activity levels were associated with high DNA fragmentation. Meanwhile, B-cell lymphoma 2 (Bcl-2) activities showed a different pattern. In conclusion, flow cytometry for sperm DNA fragmentation and markers of apoptosis can be a valuable tool in assisted reproductive centers.