Bax Expression Levels Research Articles

Oscillatory shear stress promotes vein graft intimal hyperplasia via NADPH oxidase-related pathways.

Uncontrolled intimal hyperplasia (IH) after autologous saphenous vein grafting triggers a high restenosis rate; however, its association with the activation of NADPH oxidase (NOX)-related pathways is unclear. Here, we investigated the effects and mechanism of oscillatory shear stress (OSS) on grafted vein IH. Thirty male New Zealand rabbits were randomly divided into control, high-OSS (HOSS), and low-OSS (LOSS) groups, and the vein grafts were harvested after 4 weeks. Hematoxylin and eosin staining and Masson staining assays were used to observe morphological and structural changes. Immunohistochemical staining was used to detect α-SMA, PCNA, MMP-2, and MMP-9 expression. Immunofluorescence staining was used to observe reactive oxygen species (ROS) production in the tissues. Western blotting was used to determine the expression levels of pathway-related proteins (NOX1, NOX2, AKT, p-AKT, and BIRC5), PCNA, BCL-2, BAX, and caspase-3/cleaved caspase-3 in tissues. Blood flow velocity was lower in the LOSS group than in the HOSS group, while vessel diameter did not change significantly. Shear rate was elevated in both HOSS and LOSS groups but was higher in the HOSS group. Additionally, vessel diameter increased with time in the HOSS and LOSS groups, whereas flow velocity did not. Intimal hyperplasia was significantly lower in the LOSS group than in the HOSS group. IH was dominated by smooth muscle fibers in the grafted veins and collagen fibers in the media. OSS restriction significantly reduced the α-SMA, PCNA, MMP-2, and MMP-9 levels. Moreover, ROS production and the expression of NOX1, NOX2, p-AKT, BIRC5, PCNA, BCL-2, BAX, and cleaved caspase-3 were phase-reduced in LOSS compared to the levels in the HOSS group. Total AKT was not differentially expressed among the three groups. OSS promotes the proliferation, migration, and survival of subendothelial vascular smooth muscle cells in grafted veins, which may be related to the regulation of downstream p-AKT/BIRC5 levels through the increased production of ROS by NOX. Drugs inhibiting this pathway might be used to prolong vein graft survival time.

Hepatoprotective effect of taxifolin on cyclophosphamide-induced oxidative stress, inflammation, and apoptosis in mice: Involvement of Nrf2/HO-1 signaling.

Taxifolin (TA) is a natural flavonoid found in many foods and medicinal plants with well-documented antioxidant and anti-inflammatory properties. Cyclophosphamide (CP) is an effective antineoplastic and immunosuppressive agent; however, it is associated with numerous adverse events, including hepatotoxicity. Herein, we aimed to investigate the potential protective effects of TA using a mouse model of CP-induced hepatotoxicity. Mice were co-treated with TA (25 and 50 mg/kg, orally) and CP (30 mg/kg, i.p.) for 10 consecutive days and sacrificed 24 hours later. CP induced increased transaminases (ALT and AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) paralleled with pronounced histopathological alterations in the liver. Moreover, hepatic tissues of CP-injected mice showed increased malondialdehyde (MDA), protein carbonyl, and nitric oxide (NO) levels, accompanied by decreased antioxidant defenses (glutathione [GSH], superoxide dismutase [SOD], and catalase [CAT]). Livers of CP-injected mice also showed increased inflammatory response (nuclear transcription factor kappa-B [NF-κB] p65 activation, increased levels of proinflammatory cytokines tumor necrosis factor alpha [TNF-α], interleukin 1 beta [IL-1β], and IL-6) and apoptosis (decreased Bcl-2 and increased Bax and caspase-3 expression levels). Remarkably, TA ameliorated markers of liver injury and histological damage in CP-injected mice. TA treatment also attenuated numerous markers of oxidative stress, inflammation, and apoptosis in the liver of CP-injected mice. This was accompanied by increased nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) expression in the liver tissues of CP-injected mice. Taken together, this study indicates that TA may represent a promising new avenue to prevent/treat CP-induced hepatotoxicity and perhaps other liver diseases associated with oxidative stress and inflammation.

Upregulation of KHDC1L promotes the proliferation and inhibits apoptosis in head and neck squamous cell carcinoma

ABSTRACT Head and neck squamous cell carcinoma (HNSCC) remains a dreadful malignancy bearing poor clinical efficacy, with emerging evidences indicating RNA-binding proteins’ (RBPs’) relevance to the evolution of the disease. Categorized as RBPs, the K-homology domain-containing 1 (KHDC1) family is proved to be closely related to cell survival and death. As a novel KHDC1 member, only one study is currently available in osteoarthritis synovial cells to unveil KHDC1L’s function of promoting proliferation. Nevertheless, to the best of our knowledge, the role of KHDC1L in human tumour is yet to be fully explored. On the basis of The Cancer Genome Atlas (TCGA) database and cell lines comparison with normal counterparts in this study, we first discovered KHDC1L to be overexpressed in HNSCC. According to bioinformatics analysis, apoptosis and P53 pathways were remarkably enriched in the KHDC1L low-expression group in TCGA database. Moreover, in vitro experiments were applied to verify that upregulation of KHDC1L could promote the proliferation and inhibit apoptosis in HNSCC cells CAL27. Transcriptome sequencing ascertained downstream differentially expressed genes to be significantly enriched in PI3K-AKT pathways. Furthermore, as validated by western blot, we found an elevated expression level of pAKT/AKT and Bcl-2, constant expression level of BAX, together with decreased activity of Caspase-3 and PARP-1 in the KHDC1L-upregulated group. In conclusion, our study pioneeringly elaborated that KHDC1L could promote proliferation and inhibit apoptosis in HNSCC cell CAL27 via AKT and Bcl-2 pathways, representing a crucial step for seeking a new diagnostic and therapeutic target in HNSCC.

Effects of Quercetin-3-O-β-D-Glucuronide on Platelet Apoptosis and Activation in Mice with Immune-Mediated Bone Marrow Failure via PI3K/AKT Pathway

To investigate the effect of quercetin-3-O-β-D-glucuronide(QG) on platelet apoptosis and activation in mice with immune-mediated bone marrow failure via PI3K/AKT pathway. An immune-mediated bone marrow failure mice model was established and forty C57BL/6 mice were randomly assigned into 4 groups: normal group, model group,cyclosporine (CsA) group and QG group, with 10 mice in each group.The mice in CsA group were intragastrically administered with 0.027 g/kg CsA daily and the mice in QG group were intragastrically administered with 0.2 g/kg QG daily, while the mice in the normal group and model group were intragastrically administered with normal saline, respectively. After three days of modeling, the mice were euthanized, and the blood was collected through the tail vein. Part of the blood was used for blood routine examination, and the other part was used to prepare washed platelets. Some of the prepared washed platelets were used to detect the expressions of BAX, BAD, caspase-9, phosphatidylserine (PS), platelet activated complex-1 (PAC-1) and P-selectin by flow cytometry; some of washed platelets was used to determine the protein contents of PI3K, p-PI3k, AKT and p-AKT by Western blot; the other part of the washed platelets was used to measure the expressions of PI3K mRNA and AKT mRNA by real-time quantitative PCR (RT-qPCR). In the model group, the absolute platelet count of the mice was significantly decreased, and the levels of BAX, BAD, caspase-9, PS, PAC-1, and P-selectin in the washed platelets were significantly increased, compared to the normal group (P<0.05). At the same time, the expression levels of PI3K, p-PI3K, AKT, p-AKT proteins and PI3K mRNA, AKT mRNA in model group were significantly reduced, compared to the normal group (P<0.05). In the CsA group and QG group, the expression levels of BAX, BAD, caspase-9, PS, PAC-1, and P-selectin in the washed platelets were significantly reduced (P<0.05), while the levels of PI3K, p-PI3K, AKT, p-AKT and PI3K mRNA, AKT mRNA were significantly increased, compared to the model group (P<0.05). QG can reduce platelet apoptosis in mice with immune-mediated bone marrow failure, and activation of some platelets is involved, which may be related to the regulation of PI3K/AKT pathway.

Nerolidol attenuates dehydroepiandrosterone-induced polycystic ovary syndrome in rats by regulating oxidative stress and decreasing apoptosis

AimsAlthough nerolidol (NRL) is a naturally occurring sesquiterpene alcohol with many pharmacological activities, its role in dehydroepiandrosterone DHEA-induced polycystic ovary syndrome PCOS is unknown. This study aims to explore the potential beneficial effects and underlying molecular mechanisms of nerolidol treatment on polycystic ovary syndrome. Main methodsPre-pubertal female Sprague–Dawley rats were randomly assigned into four groups (n = 8/group); group I: control; group II: PCOS; group III: P + NRL; group IV: NRL. Biochemical parameters related to oxidative stress, inflammation, apoptosis, and hormones were estimated in the blood and ovarian tissues. Histopathological, ultrastructural, and immunohistochemical analyses were performed. Bax, P53, Cas-3, and Bcl-2 gene expression levels were detected with RT-PCR. The membrane array analysis detected chemokine, cytokine, and growth factor protein profiles. Key findingsIn light of the available data, it can deduce that nerolidol has a significant ameliorating effect on lipid peroxidation, oxidative stress, inflammation, histopathological damage, and apoptosis accompanying PCOS in female rats. SignificancePCOS is not only a reproductive pathology but also a systemic condition and its etiopathogenesis is still not fully understood. Since changes in PCOS have important long-term effects on health, this study evaluated the efficacy of nerolidol, a phytotherapeutic for the control of biochemical, apoptotic, histopathological, and metabolic changes.

Myrislignan Induces Apoptosis in Gastric Cancer Cell Line Through PI3K/AKT Signaling Pathway

To investigate the effect of myrislignan (MYR) on the apoptosis of gastric cancer cell line and its relationship with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. The gastric cells (SGC-7901) were treated with MYR at different concentrations, i.e., 0, 25, 50, 100, and 200 μmol/L, for 48 h and 72 h and the effect of MYR on the proliferation of SGC-7901 cells was measured by CCK-8 assay. Then, SGC-7901 cells were treated with different concentrations of MYR at 50, 100, and 200 μmol/L for 48 h. Meanwhile, a normal control group and a dimethyl sulfoxide (DMSO) solvent control group (0.1% DMSO) were established. Flow cytometry was used to determine the apoptosis rate of SGC-7901 cells. The protein expression levels of PI3K, AKT, Bcl-2-associated X protein (BAX), cysteine-dependent aspartate-specifc protease-3 (Caspase-3), and Caspase-9 were determined by Western blot. Then, PI3K activator (20 μmol/mL) was used to treat SGC-7901 cells for 48 h in 4 groups, the control group, 0.1% DMSO group, MYR group, and MYR+PI3K activator group, and the effect on MYR's induction of apoptosis and regulation of the protein expression levels of PI3K, AKT, BAX, Caspase-3, and Caspase-9 in SGC-7901 cells. Compared with the control group, MYR at 50, 100 and 200 μmol/L inhibited the proliferation of gastric cancer cells, increased the apoptosis rate, down-regulated the protein expression levels of PI3K and AKT, and up-regulated the protein expression levels of BAX, Caspase-3, and Caspase-9 in a dose-dependent manner ( P<0.05). However, PI3K activator attenuated MYR-induced apoptosis in gastric cancer cells and MYR's regulation of PI3K, AKT, BAX, Caspase-3, and Caspase-9 protein expression ( P<0.05). MYR induces the expression of BAX, Caspase-3, and Caspase-9 proteins by inhibiting the PI3K/AKT signaling pathway, thereby promoting the apoptosis of gastric cancer cells.

Chronic stress-induced apoptosis is mitigated by young mitochondria transplantation in the prefrontal cortex of aged rats.

Apoptosis is common and often comorbid with aging and stress-related mood disorders. Evidence suggests that fresh mitochondria could reverse age-related dysfunctions in organs, especially in the brain. Therefore, this study investigated the effect of young mitochondria administration on the apoptosis process in the prefrontal cortex (PFC) of aged rats exposed to chronic stress. Aged (22 months old) male rats were randomly assigned into four groups: aged control (AC), aged rats treated with young mitochondria (A+M), aged rats subjected to chronic stress for four weeks (A+St), and aged rats subjected to chronic stress and treated with young mitochondria (A+St+M). A+M and A+St+M groups received a single ICV injection (10 μl) of fresh mitochondria isolated from the brain of young rats for five minutes (2 µl/min). Finally, the levels of Malondialdehyde (MDA), Cytochrome c (Cyt c), Bax, Bcl-2, and Caspase-3 expression were investigated in the PFC. Young mitochondria administration reduced neuronal apoptosis in the PFC, associated with down-regulation of MDA, Bax, and Caspase-3 and up-regulation of Bcl-2. Moreover, fresh mitochondria partially improved the chronic stress-induced mitochondrial dysfunction in aged rats, as indicated by reduced cytochrome c (Cyt c) release from the mitochondria. These results suggest mitotherapy could reverse cell viability and mitochondrial dysfunction-induced apoptosis in the PFC tissue of aged rats subjected to stressful stimuli.

MiR-21-5p protects against ischemic stroke by targeting IL-6R.

Ischemic stroke is a brain dysfunction disease caused by vascular obstruction. The expression of many kinds of microRNAs (miRNAs) is related to ischemic stroke. MiRNA has the ability to reduce or save ischemic injury. Therefore, we aimed to explore the protective miRNA in the ischemia-reperfusion process. The Gene Expression Omnibus (GEO) peripheral RNA sequencing (RNA-seq) datasets of ischemic stroke patients were analyzed to search for differentially expressed miRNAs in the ischemia-reperfusion process. The expression level of miRNA in 60 patients with ischemic stroke and 23 age-matched healthy control inpatients was tested by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The significantly changed miRNAs were verified through comparison of the peripheral blood of healthy people and patients of the hospital. The in-vitro ischemia-reperfusion model was established through oxygen-glucose deprivation (OGD) treated HEMC-1 cells. The cell viabilities and cell apoptosis are detected by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, respectively. Apoptosis-related proteins including Bcl-2, Bax, caspase-3, and caspase-9 expression levels were verified by western blot. Predict the combination of hsa-miR-21-5p and interleukin-6 receptor (IL-6R) through TargetScan database, clone the 2964-2961 site of IL-6R-3'-untranslated region (3'-UTR), establish IL-6R-3'-UTR and IL-6R-3'-UTR mutant plasmids, copy and clone wild type and mutant IL-6R-3'-UTR into luciferase report vector pGL3 respectively, and detect the activity of luciferase. The expression of hsa-miR-21-5p was regulated by using hsa-miR-21-5p mimic and hsa-miR-21-5p inhibitor. Through RNA-seq analysis, it was revealed that "hsa-miR-548ar-3p", "hsa-miR-651-5p", "hsa-miR-142-3p", "hsa-miR-21-5p", and "hsa-miR-30e-5p" were notably lower in ischemia patients, and that "hsa-miR-21-5p" was significantly decreased in the peripheral blood of hospital patients. Luciferase assay showed that hsa-miR-21-5p could directly bind to the 3'-UTR of the IL-6R gene and inhibit IL-6R translation; the level of IL-6R was also elevated in patients. In the OGD-treated HMEC-1 cells, overexpressed hsa-miR-21-5p mimic could enhance cell viabilities and decrease cell apoptosis. Moreover, IL-6R overexpression could reduce the protective effects of hsa-miR-21-5p. In the peripheral blood of ischemia patients, hsa-miR-21-5p is significantly decreased and IL-6R is elevated. The "hsa-miR-21-5p" could bind to the IL-6R gene and suppress IL-6R expression, thus alleviating the damage of OGD treatment in HMEC-1 cells.

Anti-Tumor Activity of the Water-Soluble Protein Extract of Tricholoma mongolicum (Agaricomycetes) in H22 Tumor-Bearing Mice and the Possible Underlying Mechanism.

Tricholoma mongolicum is a well-known edible and medicinal mushroom that is rich in protein, polysaccharides, and other nutrients and is found in China's Inner Mongolia region, which has a variety of pharmacological activities. In this study, the water-soluble protein extract of T. mongolicum (WPTM) were assessed. Further, the anti-tumor activity of the water-soluble protein extract of T. mongolicum (WPTM) in H22 tumor-bearing mice was investigated in this study. The H22 anti-tumor activity of T. mongolicum protein was studied. WPTM significantly improved interferon-γ, interleukin-2, interleukin-6, and tumor necrosis factor-α levels in serum cytokine, but decreased vascular endothelial growth factor (VEGF) levels. And WPTM treatment of H22 tumor tissues significantly increased the expression levels of BAX and caspase-3 but decreased those of Bcl-2 and VEGF in a dose-dependent manner. In summary, the findings indicate that T. mongolicum is a protein-rich edible and medicinal fungus that is a potential functional food for the prevention and treatment of liver cancer. T. mongolicum has a high protein content and nutritional value, as well as anti-tumor properties, and is expected to be widely developed.

NO-induced apoptosis in human melanocytes from lightly and darkly pigmented skin.

Nitric oxide (NO) is a potent modulator of programmed cell death, with the ability to both induce and prevent apoptosis. Some of the factors that are capable of triggering apoptosis of skin cells also cause NO overproduction in the epidermis. Unlike keratinocytes, melanin-producing melanocytes are highly resistant to apoptotic death. To investigate whether NO can induce apoptosis in normal human epidermal melanocytes and whether the pigmentation phenotype of the cells can affect their response to NO. Human epidermal melanocytes, derived from lightly and darkly pigmented neonatal foreskins, were cultured in the presence of various concentrations of SPER/NO. The effect of NO released from its donor on the cell morphology, viability, and proliferation was assessed. The ability of NO to induce cell apoptosis was evaluated by Hoechst 33342 staining, DNA fragmentation assay, flow cytometry with annexin V and propidium iodide staining, determination of caspase 3/7, 8, and 9 activities, and assessment of changes in the cell expression levels of BAX and BCL-2. We have shown that NO is capable of inducing apoptosis in normal human epidermal melanocytes in vitro, with preferential activation of the intrinsic (mitochondrial) pathway. Melanocytes from darkly pigmented skin showed a strong increase in BCL-2 expression in response to NO and were significantly more resistant to apoptosis than those from lightly pigmented skin. The pigmentation phenotype may be an important factor modulating the response of human epidermal melanocytes to proapoptotic activity of extracellular NO.

Platelet-rich plasma protects human keratinocytes from UVB-induced apoptosis by attenuating inflammatory responses and endoplasmic reticulum stress.

Although the role of platelet-rich plasma (PRP) in ultraviolet light B (UVB)-induced photoaging has been confirmed in many studies, the specific mechanism is still not clear. Therefore, we attempted to investigate the effect and mechanism of PRP on UVB-induced human keratinocyte (HaCaT cells) apoptosis. HaCaT cells were collected to construct UVB-induced photoaging models. Then, the cells were divided into Sham group, 5% PRP group, UVB group, and UVB + 5% PRP group. Next, MTT assay was used to detect the level of cell proliferation; flow cytometry to check the level of apoptosis; ELISA to determine the TNF-α, IL-18, IL-6, and IL-1β levels in the supernatant; and Western blot to test Bax, Bcl-2, cytochrome c (Cyt.c), GRP78, CHOP, and ATF4 protein expression levels. Briefly, 5% PRP intervention could relieve the inhibition of UVB on HaCaT cell proliferation, inhibit the promotion of UVB to cell apoptosis, up-regulate UVB-induced Bcl-2 protein expression, and decrease Bax and Cyt.c protein level. In addition, 5% PRP significantly down-regulated the inflammatory factor levels of TNF-α, IL-18, IL-6, and IL-1βin UVB-induced cells and reduced the inflammatory response. Moreover, 5% PRP also greatly reduced the protein expression levels of GRP78, CHOP, and ATF4 in UVB-induced cells and alleviated endoplasmic reticulum (ER) stress. PRP may protect HaCaT cells from UVB-induced apoptosis by alleviating inflammatory response and ER stress.

Erxian decoction alleviates cisplatin-induced premature ovarian failure in rats by reducing oxidation levels in ovarian granulosa cells

Ethnopharmacological relevantErxian Decoction (EXD) has been used empirically for more than 70 years to treat premature ovarian failure (POF), but more research is needed to understand how it works. Aim of the researchThe study aims to ascertain both in vivo and in vitro rewards of EXD. Materials and methodsEXD is composed of Curculiginis Rhizoma, Epimedii Folium, Morindae Officinalis, Angelicae Sinensis, Anemarrhenae Rhizoma, and Phellodendri Chinensis Cortex. UPLC/MS analysis was used to investigate the components of EXD. Using a POF model created by administering cisplatin to rats intraperitoneally, the pharmacodynamic effects of EXD were investigated. Three dose groups of EXD were garaged into rats: high (15.6 g/kg), medium (7.8 g/kg), and low (3.9 g/kg). By using a vaginal smear, the impact of EXD on the rat estrous cycle was evaluated. An ELISA test was used to measure the anti-Mullerian hormone (AMH), estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels in the serum of rats. By using HE stains, pathological alterations in the ovaries may be seen. MDA and SOD levels in ovarian samples were used to measure the degree of ovarian oxidation. TUNEL labeling of ovarian sections was used to find apoptosis levels. By using ATP, energy production was evaluated. The relative expression of proteins connected to aging and the RAGE pathway was assessed using Western blot. Then, using H2O2, a model of senescent human ovarian granulosa cells (KGN) was created in vitro. The impact of EXD and H2O2 on cellular senescence was discovered using-galactosidase staining. Cell apoptosis levels were found using PI/Hoechest33342. By using DCFH-DA, intracellular ROS was examined. MDA and SOD concentrations were used to measure the degree of cellular oxidation. RAGE-related mRNA and protein expression were evaluated using RT-qPCR and western blotting. ResultsUsing UPLC/MS analysis, 39 chemicals in EXD were found. Rats' estrous cycles were enhanced by EXD, which increased ovarian index and follicle count and reduced the proportion of atretic follicles in the rats. EXD reduced LH and FSH output while restoring AMH and E2 secretion. In ovarian tissues, EXD reduced the amount of apoptosis and MDA while raising SOD activity and ATP levels. The protein levels of p16, p21, p53, and Lamin A/C were among the senescence-related proteins that EXD lowered, along with the levels of RAGE, PI3K, BAX, and CASPASE 3. Anti-apoptotic protein BCL-2 was also raised in the RAGE pathway. Senescence, apoptosis, ROS, and MDA levels in the KGN cells were lowered in vitro by EXD. Additionally, EXD increased the anti-apoptotic potential by changing the expression of CAT, SOD2, and SIRT1. RAGE, BAX, BCL-2, CASPASE 3, and p38 expression levels were altered by EXD, enhancing its anti-apoptotic capability. ConclusionEXD boosted the ovary's antioxidant and anti-apoptotic capabilities while enhancing the estrous cycle and hormone output. These findings strongly suggested that EXD may contribute to the alleviation of POF and ovarian granulosa cells senescence.

Protective effect of multifunctional peptides PIISVYWK and FSVVPSPK on oxidative stress-mediated HUVEC injury through antioxidant and anti-apoptotic action

PIISVYWK and FSVVPSPK are antioxidant peptides identified from blue mussel protein. In the present study, the cytoprotective function of the two peptides on H2O2-mediated injuries in human umbilical vein endothelial cells (HUVECs) was evaluated. The results indicated that the peptides significantly restored cell survival (p < 0.05) in response to damage caused by H2O2 exposure. HUVECs treated with H2O2 generated high levels of ROS. While the elevated intracellular levels of ROS were decreased by the treatment with peptides; the result may be attributed to the elevated heme oxygenase-1 expression through Nrf2 nuclear translocation. Investigation of another underlying mechanism revealed that the peptides inhibited the apoptotic process in HUVECs that was induced by H2O2 exposure. Highly elevated levels of apoptotic Bax expression, the release of cytochrome C into the cytoplasm, and the cleaved caspase-3 expression were observed along with the loss of mitochondrial membrane potential (MMP). The peptides downregulated the expression of Bax and the cleaved caspase-3, inhibited the release of cytochrome C, and restored MMP in response to H2O2-mediated HUVECs injuries. In addition, the peptides increased the expression of antiapoptotic Bcl-2.

Silencing tumor-intrinsic HHLA2 potentiates the anti-tumoral effect of paclitaxel on MG63 cells: Another side of immune checkpoint

BackgroundOsteosarcoma is common type of bone cancer; however, the prognosis of patients with metastatic osteosarcoma is poor. As a new inhibitory immune checkpoint molecule, HHLA2 is upregulated in osteosarcoma. Herein, we studied the significance of tumor-intrinsic HHLA2 in MG-63 growth. Also, we examined the influence of combined therapy of HHLA2 knockdown with paclitaxel on the apoptosis, cell cycle, migration, and stemness of MG-63 cells. MethodsThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was performed to study the half-maximal inhibitory concentration (IC50) of paclitaxel and the cytotoxicity of HHLA2-small interfering RNA (siRNA) on MG-63 cells. The apoptosis and cell cycle were analyzed using flow cytometry. The wound-healing and colony formation assays were conducted to investigate the effect of paclitaxel and HHLA2 knockdown on the migration and stemness of MG-63 cells, respectively. QRT-PCR was used to determine the Bax, caspase-3, and Bcl-2 mRNA expression levels. ResultsHHLA2 silencing has enhanced the chemosensitivity of MG-63 cells to paclitaxel. Besides, HHLA2 knockdown has increased the paclitaxel-induced cytotoxic effect on MG-63 cells. In terms of stimulating apoptosis, decreasing clonogenicity, halting the cell cycle at the sub G1 phase, and inhibiting migration, tumor-intrinsic HHLA2 silencing has increased these anti-tumor effects of paclitaxel on MG-63 cells. Besides, HHLA2 knockdown has potentiated paclitaxel-mediated Bcl-2 downregulation and paclitaxel-mediated caspase-3 and Bax upregulation in MG-63 cells. ConclusionTumor-intrinsic HHLA2 knockdown increases the anti-tumoral effect of paclitaxel on MG-63 cells and enhances the chemosensitivity of MG-63 cells to paclitaxel.

Design, Synthesis, Docking Studies and Biological Evaluation of Novel Benzochromenopyrimidines via Silica Sulfuric Acid Catalyzed Reaction on Apoptosis in Human Cancer Cells

Via one-pot reaction of 2-naphthol, thiobarbituric acid and aldehyde in the presence of silica sulfuric acid (SSA) as a catalyst to synthesis benzochromenopyrimidines derivatives. Then we studied the reaction of benzochromenopyrimidines derivatives with bromoacetic acid to obtain benzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidine-11,13(10H,14H)-dione derivatives. Meanwhile, dichloroethane was reacted with 12-(4-fluorophenyl)-9-thioxo-9,10-dihydro-8H-benzo[5,6]chromeno[2,3-d]pyrimidin-11(12H)-one to form 14-(4-fluorophenyl)-10,11-dihydrobenzo[5,6]chromeno[2,3-d]thiazolo[3,2-a]pyrimidin-13(14H)-one. All new products structure was elucidated based on analytical and spectroscopic analyses. Anti-cancer activity of newly synthesized structures was investigated. Cancer cells (MDA-MB-231 and HepG2) were used to evaluate the anti-cancer activity of tested compounds using neutral red uptake assay. Treatment of MDA-MB-231 and HepG2 cells with tested compound 6h revealed more inhibitory influence after 48 h. The expression levels of BCL2, BAX, Caspase3, Survivin and P53 genes were investigated using QRT-PCR. This study exhibited that compound 6h revealed significant pro-apoptotic effect via down regulation of BCL2 and Survivin genes and up-regulation of BAX, Caspase3 and P53 genes when treated with MDA-MB-231 and HepG2 cells as compared to control values. The biological studies of these compounds were proved through molecular docking studies with human cyclin-dependent kinase 2 (PDB code: 2A4L) and compound 6h showed low binding energy and shortage bond length with different amino acids. Noteworthy, the tested compound 6h exhibited the most pronounced effect in this respect.

Honokiol Reduces Mitochondrial Dysfunction and Inhibits Apoptosis of Nerve Cells in Rats with Traumatic Brain Injury by Activating the Mitochondrial Unfolded Protein Response.

This study was designed to determine the effects and underlying mechanism of honokiol (HNK) on traumatic brain injury (TBI). A rat TBI model was constructed using the modified Feeney free-fall percussion method and treatment with HNK via intraperitoneal injection. The brain tissues of the rats in each group were assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay to detect the level of neuronal apoptosis. Western blots were used to detect the expression levels of apoptosis-related proteins (Bcl-2 and Bax), and ELISAs were used to measure the levels of pro-inflammatory cytokines (IL-18 and IL-1β) and the activity of caspase-1. In addition, the mitochondrial membrane potential, reactive oxygen species (ROS), and adenosine 5'-triphosphate (ATP) were also measured. Western blots and qRT-PCRs were used to determine the relative expression levels of the mitochondrial unfolded protein response (UPRmt)-related proteins and mRNAs. Based on the experimental results, treatment with HNK was associated with a decrease in the number of TUNEL-positive cells, downregulated Bax expression levels, elevated Bcl-2 expression levels, and inhibition of neuronal apoptosis in the brain tissue of TBI rats. HNK also suppressed neuroinflammation by decreasing IL-1β and IL-18 levels and caspase-1 activity. Additionally, HNK lowered the mitochondrial membrane potential and ROS levels, increased ATP levels, and improved mitochondrial dysfunction in neural cells. Furthermore, in the investigation of the mechanism of HNK on TBI, we observed that HNK could activate UPRmt by upregulating the mRNA and protein expression levels of HSPA9, CLPP, and HSP60 in the brain tissues of TBI rats. Collectively, HNK reduced mitochondrial dysfunction, inhibited the apoptosis of nerve cells, and attenuated inflammation in the brains of TBI rats. The protective effect of HNK may be achieved through the activation of UPRmt.

Decreased thioredoxin reductase 3 expression promotes nickel-induced damage to cardiac tissue via activating oxidative stress-induced apoptosis and inflammation.

Thioredoxin reductase 3 (Txnrd3) plays a crucial role in antioxidant and anti-cancer activities, and sperm maturation. The damage of heavy metals, including Nickel (Ni), is the most prominent harm in social development, and hampering Txnrd3 might exacerbate Ni-induced cardiac damage. In this study, a total of 160 8-week-old C57BL/N male mice with 25-30 g weight of Txnrd3+/+ wild-type and Txnrd3-/- homozygote-type were randomly divided into eight groups. The mice in the control and Ni groups were gavaged with distilled water and a freshly prepared 10mg/kg NiCl2 solution. Melatonin (Mel) groups were administered at a concentration of 2mg/kg for 21 days at the mice's 0.1ml/10g body weight. Ni exposure up-regulated the messenger RNA (mRNA) levels of mitochondrial apoptosis (caspase-3, caspase-9, cytochrome c, p53, and BAX), autophagy (LC3, ATG 1, ATG 7, and Beclin-1), and inflammation (TNF-α, COX 2, IL-1β, IL-2, IL-6, and IL-7)-related markers, but down-regulated the mRNA levels of BCL-2, p62 and mTOR (p < .05). Ni exposure decreased the expression of BCL-2 and p62 protein but increased the expression levels of caspase-3, caspase-9, cytochrome c, p53, BAX, ATG 7, Beclin-1, TNF-α, COX 2, IL-1β and IL-2 protein (p < .05). Ni increased the contents of glutathione disulfide (GSSG) and malondialdehyde (MDA) and decreased the activities of catalase (CAT) and total superoxide dismutase (T-SOD) (p < .05). Decreased Txnrd3 expression significantly exacerbated changes compared to the Ni exposure (p < .05). Mel significantly attenuated these changes, but the effect decreased when Txnrd3 was inhibited (p < .05). In conclusion, decreased Txnrd3 expression promoted Ni-induced mitochondrial apoptosis and inflammation via oxidative stress and aggravated heart damage in mice. Decreased Txnrd3 expression significantly reduced the protective effect of Mel to Ni exposure.

ΔNp63α transcriptionally represses p53 target genes involved in the radiation-induced DNA damage response

BackgroundThe DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and ΔNp63α, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. ΔNp63α has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of ΔNp63α on radiation-induced DDR.MethodsTo elucidate the role of the p53-repressive effect of ΔNp63α in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing ΔNp63α and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in ΔNp63α-expressing and control cells was analysed by RT–qPCR, Western blotting, and flow cytometry.ResultsThe mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2´-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of ΔNp63α in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation.ConclusionTaken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells.

DNA and RNA Profiles in Machine Learning Algorithm to Predict Which Patients with AML/MDS Will Respond to Venetoclax-Based Therapy

Introduction Venetoclax in combination with hypomethylation agents is currently used as an alternative to standard induction therapy in patients with acute myeloid leukemia (AML) or advanced myelodysplastic syndrome (MDS). This is particularly relevant when patients are not eligible for standard induction therapy due to age or comorbidity. However, little is known about which patients will benefit from such therapy and which patients will not respond. Selecting the right patient for such therapy is very important considering the clinical fragility of these patients. Genomic profiling using DNA and RNA is becoming a standard of care in clinical testing and there is a need to expand on the clinical utility of the generated data for this profiling. We explored the potential of using these genomic DNA and RNA profiles in a machine learning algorithm to predict which patients with AML/MDS will respond to venetoclax-based therapy. Methods: DNA and RNA from bone marrow samples from 46 patients with AML or advanced high-grade MDS treated with venetoclax (plus either azacitidine or decitabine) were extracted. Using next generation sequencing (NGS), the DNA was sequenced using a 177-gene panel and the RNA was sequenced using a 1408-gene panel. Sequencing was performed using the Illumina NextSeq 550 platform. Ten million reads per sample in a single run were required. Read length was 2 × 150 bp. Expression profiles were generated using Cufflinks. We developed a machine learning algorithm that first selected the relative genes based on performance of each gene with cross-validation and based on stability measures using statistical significance tests. The selected genes were then used to predict response or survival with a k-fold cross-validation procedure (k=12). A naïve Bayesian classifier was constructed on the training of k-1 subsets and tested on the other testing subset. We applied geometric mean naïve Bayesian (GMNB) as the classifier for prediction. Results: Of the 46 patients included in the study, 18 (39%) were females. The median age was 70.5 years (range 33-84). The most common mutations detected in these patients were: ASXL1 (41%), RUNX1 (26%), DNMT3A (24%), FLT3 (21%), NPM1 (20%), NRAS (20%), IDH1/2 (20%), TET2 (17%), TP53 (15%), and SRSF2 (11%). Of these 46 patients, 17 (37%) achieved complete response (CR). Using machine learning we were able to predict those who achieved CR with AUC of 0.972 (95% CI: 0.914-1.00) (Figure), sensitivity of 94.1% and specificity of 93.1%. To achieve this prediction only 10 genes were necessary. There was no significant difference between responders and non-responders in expression level of BCL2 (P=0.11), BAX (P=0.18), or MCL1 (P=0.54). BCL2L1 (BCL-XL) was significantly higher in non-responders (P=0.006) as compared with responders. TP53 was mutated in seven patients, six of whom were non-responders, but no other statistically significant association between specific mutation and response occurred. With a median follow-up of 13 months, 28 (61%) patients were alive and 18 (39%) were dead. Using a machine learning algorithm, we were able to predict overall survival with AUC of 0.970 (95% CI: 0.923-1.00 ) (Figure), sensitivity of 100% and specificity of 94.4%. The algorithm selected 90 genes for this prediction. Conclusion: This data shows that bone marrow expression profiling when used in machine learning can provide a valuable and practical approach for predicting response and overall survival to venetoclax-based therapy in patients with AML/MDS. This prediction requires complex data from a large number of genes (10 to 90 genes) that must be incorporated in a machine learning algorithm. While further studies are needed for confirmation, genomic profiling along with machine learning algorithms can be a practical test for selecting a specific therapeutic approach for specific patients with AML/MDS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Design, Synthesis, Molecular Modeling, and Anticancer Evaluation of New VEGFR-2 Inhibitors Based on the Indolin-2-One Scaffold.

A new series of indoline-2-one derivatives was designed and synthesized based on the essential pharmacophoric features of VEGFR-2 inhibitors. Anti-proliferative activities were assessed for all derivatives against breast (MCF-7) and liver (HepG2) cancer cell lines, using sunitinib as a reference agent. The most potent anti-proliferative derivatives were evaluated for their VEGFR-2 inhibition activity. The effects of the most potent inhibitor, 17a, on cell cycle, apoptosis, and expression of apoptotic markers (caspase-3&-9, BAX, and Bcl-2) were studied. Molecular modeling studies, such as docking simulations, physicochemical properties prediction, and pharmacokinetic profiling were performed. The results revealed that derivatives 5b, 10e, 10g, 15a, and 17a exhibited potent anticancer activities with IC50 values from 0.74-4.62 µM against MCF-7 cell line (sunitinib IC50 = 4.77 µM) and from 1.13-8.81 µM against HepG2 cell line (sunitinib IC50 = 2.23 µM). Furthermore, these compounds displayed potent VEGFR-2 inhibitory activities with IC50 values of 0.160, 0.358, 0.087, 0.180, and 0.078 µM, respectively (sunitinib IC50 = 0.139 µM). Cell cycle analysis demonstrated the ability of 17a to induce a cell cycle arrest of the HepG2 cells at the S phase and increase the total apoptosis by 3.5-fold. Moreover, 17a upregulated the expression levels of apoptotic markers caspase-3 and -9 by 6.9-fold and 3.7-fold, respectively. In addition, 17a increased the expression level of BAX by 2.7-fold while decreasing the expression level of Bcl-2 by 1.9-fold. The molecular docking simulations displayed enhanced binding interactions and similar placement as sunitinib inside the active pocket of VEGFR-2. The molecular modeling calculations showed that all the test compounds were in accordance with Lipinski and Veber rules for oral bioavailability and had promising drug-likeness behavior.