Dependent Apoptotic Pathway Research Articles

Silica nanoparticles induce male reproductive toxicity via Crem hypermethylation mediated spermatocyte apoptosis and sperm flagella damage.

Exposure to silica nanoparticles (SiNPs) could causally contribute to malfunctioning of the spermatogenesis, but the underlying mechanism is rarely known. This study was designed to explore the mechanism of Crem hypermethylation in SiNP-induced reproductive toxicity. The male mice were exposure to SiNPs (0 and 20mg/kg·bw) once every 5days via intratracheal instillation for 35days. After exposure stopped, half of each group was killed, and the rest were sacrificed after another 15-day feeding. GC-2 cells were treated with 0 and 20μg/mL SiNPs. The results showed that SiNPs led to structure damage of spermatocyte and sperm, caused spermatocyte apoptosis, and decreased sperm quantity and quality. After 15days of the withdrawal, the testicular tissue damage gradually recovered. Mechanistic study showed that SiNPs induced hypermethylation of the gene of cAMP responsive element modulator (Crem) in the promoter region. Downregulation of Crem inhibited the expression of outer dense fiber 1 (Odf1), resulting in abnormal sperm flagella structure; at the same time, Crem inhibited the expression of Bcl-xl, causing upregulation of cytochrome-C, cleaved-caspase-9/caspase-9, cleaved-caspase-3/caspase-3, resulting in mitochondrial dependent apoptotic pathway. However, 5-aza, DNA methylation inhibitor, could reverse the SiNP-induced downregulation of Crem and reverse the Crem/Bcl-xl-mediated mitochondrial dependent apoptotic pathway. These results suggested SiNPs could disrupt spermatogenesis by causing Crem hypermethylation to regulate the Odf1 and Bcl-xl in spermatocytes resulting in the sperm flagella structure and spermatocyte apoptosis. Our study provided new insights into the male reproductive toxicity mechanism of SiNPs; Crem demethylation may be a potential way to prevent reproductive dysfunction from SiNP exposure.

Effective seed cake extract of O. sanctum inducing the p53 dependent apoptotic pathway in oral cancer cells

Objective Present study was to observe the therapeutic aspects of seed cake extracts of Ocimum sanctum against the oral cancer cell line with the activation of p53 apoptotic pathway. Method Seed cake extracts were characterized using GC-MS analysis. Cytotoxic activity was observed on KB cells and L929 cell through MTT assay and scratch assay. Antioxidant activity on KB cells were determined using enzymatic and non enzyme content in the treated cells. Chick chorioallantoic membrane (CAM) was established to check the presence of blood vessel formation and neuvasculature pattern in the treated fertilized eggs. DNA fragmentation and gene expression studies were also determined in the treated cells to check the upregulation of apoptotic pathways. Results GC-MS analysis confirmed alkaloids, phenols, and many. The cytotoxic activity showed maximum antiproliferative potential with aqueous extract, whereas no cytotoxic effect was observed on L929 cells. The ethanolic and aqueous extract has shown a greater SI value. Scratch assay has signified that aqueous extract has a lower migration rate of KB cells. Aqueous extract showed maximum enzymatic activity and lower malondialdehyde content in cells treated with ethanolic extract. CAM model confirmed that eggs treated with aqueous extract has shown inhibition of vasculature pattern and dissolutions of blood vessels. DNA Fragmentation and Gene expression studies confirmed maximum fold in the KB cell treated with an aqueous extract of seed cake leading to activation of p53 dependent apoptotic pathway. Conclusion The potent therapeutic properties of seed cake extracts have been proven, and they can be used as herbal treatments to prevent oral cancer.

In vitro anti-proliferative, and in silico ribonucleotide reductase and pharmacokinetics studies of heteroleptic silver(I), nickel(II) and copper(II) complexes of 4-methyl-3-thiosemicarbazones and ibuprofen

ObjectivesThe present research focuses on the in vitro anti-proliferative, and in silico ribonucleotide reductase and pharmacokinetics studies of twelve heteroleptic metal complexes of the general formulae [Ag(L1−4)(ibu)] (1–4) and [M(L1−4)(ibu)2] (5–12), where L1−4 = 2-(1-(4-substitutedphenyl)ethylidene)-N-methylhydrazinecarbothioamide, ibu = non-steroidal anti-inflammatory drug (ibuprofen), and M = Cu(II) and Ni(II). MethodsVarious spectroscopic techniques were used to authenticate the structure of the synthesized complexes. UV-Vis and cyclic voltammetry techniques were used to analyse the stability and the reducing ability of the complexes. In vitro anti-proliferative studies by MTT assay, apoptotic behaviour and cellular uptake studies were investigated followed by the in silico interaction with ribonucleotide reductase (RNR) enzyme. ResultsThe spectral studies predicted distorted tetrahedral geometry around silver(I) ion and distorted octahedral geometry around nickel(II) and copper(II) ions. The reducing ability of the copper(II) complexes was analysed using ascorbic acid by UV-Vis and cyclic voltammetry techniques, which authenticate the reducing ability of the complexes and the possible interactions within the cells. The in vitro anti-proliferative activity of the synthesized complexes against three cancerous (estrogen positive (MCF-7), estrogen negative (MDA-MB-231) and pancreatic (PANC-1)) and one normal (MCF-10a) cell lines by MTT assay showed enhanced activity for copper(II) complexes 11 and 12 containing the hydrophobic substituents. The apoptotic and cellular uptake studies showed that the complex 12 is readily taken up by PANC-1 cell lines and induces ROS-mediated mitochondrial and caspase-dependent apoptosis. The in silico studies indicated hydrogen bonding, hydrophobic and π-pair (π–π, π–σ and π–cation) interactions between the complexes and the ribonucleotide reductase (RNR) enzyme. The in silico pharmacokinetics studies of the complexes predicted the drug-likeness characteristics of the complexes. ConclusionThe synthesized complexes are found to be less toxic to normal cells and inhibit the growth of cancerous cells by inducing mitochondrial-mediated and caspase dependent apoptotic pathway in PANC-1 cells.

Methanol extract of Pergularia daemia (Forssk.) Chiov. leaves induce apoptosis in triple-negative breast cancer through intrinsic pathway

Most traditional anticancer medications are ineffective in treating triple negative breast cancer (TNBC), which has enhanced resistance to chemotherapeutic agents. Herbs continue to be a key element in the discovery of anticancer medications. In the present study, we assessed the cytotoxicity of methanol extract of leaves of Pergularia daemia (MLPD) in MDA-MB-231 triple-negative breast cancer (TNBC) cell line. The cytotoxicity of MLPD was assessed using MTT assay which revealed significant concentration-dependent cytotoxicity with an IC50 value of 35.95±3.57 µg/mL. Morphological evaluation of MDA-MB-231 cells treated with MLPD showed cytotoxic changes like vacuole formation, altered morphology, and reduction in the number of cells with apoptotic bodies formation. Staining techniques, such as acridine orange and ethidium bromide (AO/EB) showed early apoptosis characterized by yellow-green fluorescence and crescent-shaped nucleus. Hoechst 33258 staining of MDA-MB-231 cells treated with MLPD showed nuclear marginalization indicative of nuclear apoptotic changes. To assess the mitochondrial membrane potential (MMP), we did JC-1 staining and the results revealed green fluorescence indicative of mitochondrial dependent apoptotic pathway. Comet assay confirmed significant DNA damaging property in MDA-MB-231 cells treated with MLPD. Western blotting analysis showed significant downregulation of Bcl-2 by 0.52±0.03 folds with no significant change in caspase-8 expression. Hence, the present study demonstrated that MLPD possessed potent cytotoxic effect against MDA-MB-231 TNBC cells through mitochondria mediated intrinsic pathway of apoptosis.

Novel pathological findings and immunohistochemical detection of FMDV antigens in the brain of calves naturally infected with foot-and-mouth disease

Foot-and-mouth disease (FMD) is an extremely contagious and economically devastating viral disease of cloven-hoofed domestic and wildlife animals. The disease is endemic in India and other developing countries of the world. The disease is mainly characterized by the presence of vesicular lesions and “tigroid heart” in calves. The current report describes the novel pathologic findings along with the distribution of FMDV antigens in brain of young calves naturally infected with FMDV. The carcasses of 37 calves suspected to have died from FMD were presented for postmortem investigation. Out of 37 dead calves, 10 calves showed the clinical signs of neurological abnormalities like opisthotonos, muscle twitching and tremor in hind limbs, stiffening of the neck followed by death. Microscopically, the meninges were congested, hemorrhagic, and infiltrated with mononuclear cells. The various sub anatomical sites of the brain showed the varying degrees of vascular changes, perivascular cuffing, focal to diffuse gliosis as well as degeneration and neuronal necrosis, indicating the nonsuppurative encephalitis. The immunolabeling of FMDV antigen was demonstrated in the neurons, inflammatory cells, and microglial cells besides its typical locations. The neurons of the brain also showed strong immunopositivity for caspase-3, caspase-9 and p53 and negative for Bcl-2 and apoptosis-inducing factor (AIF) by both immunohistochemistry and western blotting indicating the role of caspase mediated intrinsic, and p53 dependent apoptotic pathway. Further, the TUNEL assay also confirmed the apoptosis in the neurons and glial cells of the brain of naturally infected calves. This study in calves establishes a basis for resemblance to other members of Picornaviruses, such as Enterovirus 71 and Coxsackievirus of humans and showing the neuropathological alterations along with the distribution of FMDV antigens associated with apoptosis in younger calves.

Apoptosis Triggering, an Important Way for Natural Products From Herbal Medicines to Treat Pancreatic Cancers.

Pancreatic cancer, a poor prognosis and high morbidity and mortality cancer, is a malignant tumor occurring in pancreatic exocrine glands. Currently, surgery and gemcitabine (Gem) are commonly used to treat pancreatic cancers. However, the high recurrence rate and resistance makes the therapeutic effects still unsatisfied. Apoptosis is comprehensively recognized as one of the major ways of the programmed cell death, refers to the autonomous and orderly death process of cells in order to maintain the stability of the body's environment after receiving a certain signal or stimulation. Currently, it has also been proven to be a promising way for the treatment of pancreatic cancer. Nowadays, some active ingredients from herbal medicine have been reported to be effective for the treatment of pancreatic cancer via inducing cells apoptosis. Therefore, this article reviews the current references regarding anti pancreatic cancer effects of natural products derived from herbal medicines via triggering apoptosis, and summarizes the related potential signal pathways, including death receptors mediated apoptotic pathway, mitochondrial dependent apoptotic pathway, NF-κB mediated apoptotic pathways, MAPK mediated apoptotic pathway, ERS mediated apoptotic pathway, PI3K-Akt mediated apoptotic pathway, and other pathways such as JAK-STAT signal pathway, which can lay a certain foundation for the research and development of new natural products against pancreatic cancer.

Chemical Profiling and Evaluation of Antioxidant and Anticancer Potential of Tuber Crop Amorphophallus commutatus var. wayanadensis.

Cancer and cancer-related diseases are a global health concern in the present scenario. Functional food and nutraceuticals are considered as a boon towards cancer management. Amorphophallus commutatus var. wayanadensis (ACW) is an herbaceous plant used by the local communities of Wayanad, India, for food and primary healthcare. Various radical scavenging and reducing power assays were undertaken to evaluate the antioxidant activity of methanolic extract of ACW (MEAC). In vitro anticancer activity was evaluated against HT-29 cell line by MTT assay, morphological analysis, DNA fragmentation assay and cell cycle analysis. Caspase and COX-2 enzyme assays were conducted to examine the underlying mechanism. Studies on Ehrlich Ascites Carcinoma (EAC) transplanted mice models was carried out to evaluate the in-vivo antioxidant and anticancer potential of MEAC. The major bioactive nutraceutical compound present in MEAC was isolated by bioactivity-guided fractionation. MEAC showed significant in vitro antioxidant activity. Further, MEAC promoted cytotoxicity against HT-29 cells by activating caspase-3 dependent apoptotic pathway with a cell cycle arrest at the G1/S phase and subsequent down regulation of COX-2 pathway. The potential antitumor activity of MEAC was further confirmed in EAC tumor bearing mice models in which treatment with MEAC increased the levels of antioxidant enzymes, improved the hematological profile towards normal and also augmented the life span of tumor bearing mice. β-sitosterol isolated from ACW induces anticancer activity via caspase-dependent pathway. Our study confirmed the antioxidant and anticancer activities of ACW, which proposes the medicinal importance of this plant as a preventive and supportive therapy for arising tumors.

Bisphenol A induced apoptosis via oxidative stress generation involved Nrf2/HO-1 pathway and mitochondrial dependent pathways in human retinal pigment epithelium (ARPE-19) cells.

Bisphenol A (BPA) is an estrogen-like compound, and an environmental hormone, that is commonly used in daily life. Therefore, it may enter the human body through food or direct contact, causing BPA residues in blood and urine. Because most studies focused on the analysis of BPA in reproductive cells or tissues, regarding evidence the effect of BPA on human retinal pigment epithelium (ARPE-19) cells unavailable. Accordingly, the present study explored the cytotoxicity of BPA on ARPE-19 cells. After BPA treatment, the expression of Bcl-XL an antiapoptotic protein, in the mitochondria decreased, and the expression of Bax, a proapoptotic protein increased. Then the mitochondrial membrane potential was affected. BPA changed in mitochondrial membrane potential led to the release of cytochrome C, which activated caspase-9 to promote downstream caspase-3 leading to cytotoxicity. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase 1 (HO-1) pathway play a major role in age-related macular degeneration. Our results showed that expression of HO-1 and Nrf2 suppressed by BPA. Superoxide dismutase and catalase, which Nrf2 downstream antioxidants, were degraded by BPA. AMP-activated kinase (AMPK), which can regulate the phosphorylation of Nrf2, and the phosphorylation of AMPK expression was reduced by BPA. Finally, BPA-induced ROS generation and cytotoxicity were reduced by N-acetyl-l-cysteine. Taken together, these results suggest that BPA induced ARPE-19 cells via oxidative stress, which was associated with down regulated Nrf2/HO-1 pathway, and the mitochondria dependent apoptotic signaling pathway.

Autophagy triggers endoplasmic reticulum stress and C/EBP homologous protein-mediated apoptosis in OGD/R-treated neurons in a caspase-12-independent manner

We reported that a high level of autophagy was initiated by oxygen-glucose deprivation (OGD) and was maintained in neurons even after oxygen-glucose deprivation followed by reoxygenation (OGD/R), accompanied by neuronal apoptosis. This study focused on autophagy-induced apoptosis and its signaling network, especially the role of endoplasmic reticulum stress (ERS). Analysis of primary cultured cortical neurons from mice showed that the autophagy-induced apoptosis depended on caspase-8 and -9 but not on caspase-12. This finding did not mean that the endoplasmic reticulum did not participate in this process. Increases in the levels of endoplasmic reticulum (ER) biomarkers and binding immunoglobulin protein (BiP) were induced by autophagy in OGD/R-treated neurons. In addition, as an apoptotic transcription factor induced by ER stress, C/EBP homologous protein (CHOP) expression was significantly increased in neurons after OGD/R. This result suggested that the autophagy-BiP-CHOP-caspase (8 and 9)-dependent apoptotic signaling pathway at least partly participated in autophagy-induced apoptosis in primary cortical neurons. It revealed that ER induced apoptosis in neurons suffering from OGD/R injury in an ER stress-CHOP-dependent manner rather than a caspase-12-dependent manner. However, more research on signaling or cross-linking networks and intermediate links is needed. The realization of caspase-12-independent BiP-CHOP neuronal apoptosis pathway has expanded our understanding of the neuronal apoptosis network, which may eventually provide endogenous interventional strategies for OGD/R injury after stroke.NEW & NOTEWORTHY ER stress induced by autophagy mediates caspase-8- and caspase-9-dependent apoptosis pathways by regulating CHOP in neurons exposed to OGD/R. We hypothesized that the autophagy-BiP-CHOP-caspase (8 and 9)-dependent apoptotic signaling pathway at least partly participated in autophagy-induced apoptosis in primary cortical neurons.

Studies on cancer cell death through delivery of dopamine as anti-cancer drug by a newly functionalized cobalt ferrite nano-carrier

Recently, a promising applicability of dopamine (DA) as an anticancer drug has been observed because it has an anti-angiogenesis property regulating tumour growth. We have proposed here a new approach for cancer therapy using cobalt-ferrite (CF) nanoparticles as a DA delivery agent by functionalizing CF with DA and polyethylene glycol (PEG). CF was synthesized by simple wet chemical method and its physical properties were investigated by X-ray diffraction (XRD) and Transmission Electron Microscopy (TEM) to confirm its structural and morphological properties respectively. After that DA was conjugated with CF (CF-DA) and then the CF-DA was polyethylene glycolated (CF-DA-PEG) for better fixing of DA on the CF and to deliver the dopamine very slowly into the cancer cells. FTIR study confirms the adsorption of DA and coating of PEG. We then carried on some experiments on cancer cells treating them with CF, DA, CF-DA, PEG and CF-DA-PEG to investigate and compare their anticancer properties. Detail mechanisms of cancer cell death were investigated by different techniques such as MTT assay, colony formation assay, anti migratory property, cell apoptosis, reactive oxygen species (ROS) generation, actin cytoskeleton disruption, western blot analysis, mitochondrial membrane disruption investigation etc. Our results convey an important message that CF-DA-PEG is able to deliver DA inside the A549 cells and also CF-DA-PEG shows an anti-cancer effect inducing apoptosis through activating the cyrochrome-c and caspase dependent apoptotic pathway and CF catalysed ROS generation.

Low energy shock wave therapy attenuates mitochondrial dysfunction and improves bladder function in HCl induced cystitis in rats

BackgroundWe examine the effects of low energy shock wave (LESW) on bladder and mitochondrial function in a rat model of HCl induced cystitis, and the influence of dynamic bladder filling volume on LESW responses. Dysregulation of mitochondria function may impact the urothelial barrier and contribute to bladder dysfunction in patients with Interstitial cystitis/bladder pain syndrome (IC/BPS). MethodsFemale Sprague–Dawley rats underwent urethral catheterization and intravesical instillation of 0.2 ml of 0.4 N HCl (N = 32) or 0.2 ml saline (N = 8) kept for 90 s. After HCl instillation, the bladder received LESW treatment while filled with 0 ml, 0.2 ml or 0.4 ml saline or no LESW treatment. Continuous cystometry (CMG) was performed on day 8. The bladder was harvested after CMG for histology and Western blotting. ResultsHCl provoked bladder overactivity, bladder wall inflammation marked by infiltration of mast cells, increased bax/bcl2 ratio consistent with increased TUNEL staining and increased release of mitochondrial-integrity markers (cleaved caspase 3 and Cytochrome c). LESW treatment suppressed HCl provoked bladder overactivity in association with lower inflammatory reaction, mast cells infiltration, and a lower bax/bcl2 ratio also reflected by reduced TUNEL staining and mitochondrial-integrity markers irrespective of the volume of saline in bladder at the time of LESW. ConclusionsThese findings support that antiinflammatory effect of LESW in chemical cystitis is associated with the reversal of the molecular-cellular perturbations in mitochondrial dependent intrinsic apoptotic pathway.

Cyclotrisiloxan and β-Sitosterol rich Cassia alata (L.) flower inhibit HT-115 human colon cancer cell growth via mitochondrial dependent apoptotic stimulation.

Cancer traits dependent chemo and radiotherapy display acute toxicity and long-term side effects. Since last two decades, researchers investigated a new anticancer agents derived from plants. Cassia alata (L.) is a medicinal herb distributed in the tropical and humid regions. In this study, C. alata flower methanol extract (CME) have been prepared using cold percolation method and the phytochemical components were identified using GC–MS analysis. CME have been used to study the antiproliferative and apoptosis properties against human colon cancer HT-115 colon cancer cells, its molecular mechanism have been explored. 0.2 mg/mL dose of CME, inhibited 50% of HT-115 colon cancer cell growth after 48hr was confirmed the significant antiproliferation effect. In normal cells such as Vero cells and hMSCs, 0.2 mg/mL dose of CME shown only 4% and 5% growth inhibition confirmed the HT-115 cell specific cytotoxic effect. This effect might be due to the availability of phytoactive biomolecules in CME such as, cyclotrisiloxan, beta-sitosterol and alpha-tocopherol have been confirmed by GC–MS. Most interestingly, PI and AO/ErBr staining of CME treated HT-115 cells shown early (25%), pro (17%) and late (8%) apoptotic and 3% necrotic cells after 48 hr. Treatment with CME extract showed potential effect on the inhibition of protumorigenic inflammatory and oxidative stress genes. Protumorigenic COX-2/PGE-2 and TNF-α/NF-κB immune axis were normalized after CME treatment. Amounts of both apoptosis related mRNA p53, Bax, caspase 3 and p21 genes were upregulated, whereas it resulted in significant reduction in the anti-apoptotic marker mdm2 and Bcl-2 genes. In conclusion, bioactive compounds present in CME potentially inhibit HT-115 colon cancer cell proliferation via an inhibition of protumorigenic immune axis and stimulation of mitochondria dependent apoptotic pathway without necrotic effect.

Halofuginone Exerts Antileukemic Effects in TP53 Mutated Cell Lines

Introduction: The application of the standardized therapy based on anthracyclines and cytarabine for all patients with AML regardless of which molecular aberration is present, results in a heterogeneous response exemplified by a worse prognosis for both young and elderly patients exhibiting TP53 mutation. Thus, the treatment of AML requires adjustments in order to improve the patient's therapeutic response. Recently, a new compound - Halofuginone (HF), revealing multitarget effects (antifibrotic, anti-inflammatory and pro-apoptotic) was described as a potential anticancerogenic drug. The diverse targets of HF have been studied in several tumors such as in multiple myeloma, where HF triggers the caspase dependent apoptotic pathway. Our group conducted a study in APL cell lines (NB4 and NB4-R2 - resistant to ATRA treatment), demonstrating HF anti-proliferative and anti-angiogenic effects in vitro suggesting that this molecule may become a new therapeutic agent.Objective: Investigating the effect of HF in AML cell lines and characterize its impact on cellular signaling and function.Methods: The AML cell lines (Kasumi-1, U937, OCI-AML3 and THP-1) were treated with HF in ascending concentrations (50-1000 nM) for 24 hours and the cell viability, apoptosis (annexin-V/PI and caspase 3/PARP1 cleavage) and cell cycle (flow cytometry) was evaluated. The expression of apoptosis related genes (BIRC5, BCL2, BAX and TNFR) and proteins was assessed by qPCR and western blot respectively, while the presence of phosphorylated proteins involved in signaling pathways was determined by the Proteome Profiler TM Array - HumanPhospho-Kinase Array. All experiments were performed using HF at the concentration of 125 nM at 3, 6, 12 and 24 hours. To study the anti-leukemic effect in vivo, NSG mice (n=16) were transplanted with either Kasumi-1 or THP-1 cells via mouse tail vein injection and subsequently treated intraperitoneally with HF 150 mg/kg/day for 14 days and followed for overall survival (OS) analysis. Statistical analyses were performed by Student's t-test or Mann-Whitnney test, as appropriate. For survival analyses, Kaplan-Meier method was applied and log-rank test was used to compare the curves.Results: The cell viability of all cell lines was reduced upon HF treatment in a dose-dependent manner. Performing a nonlinear regression analysis, the IC50 for cytotoxicity was determined to be 125, 155, 282 and 323 nM for Kasumi-1, U937, OCI-AML3, and THP-1, respectively. Based on this data we decided to study the effects of HF in Kasumi-1 (HF sensitive cell line with a p53 p.R248Q mutation but with normal protein levels) and THP-1 (HF resistant cell line with a p53 p.R174fs) using a single HF concentration based on the IC50 value. The administration of HF in Kasumi-1 and THP-1 resulted in a decrease of S-phase cells for Kasumi-1 after 24h (P=.004). HF treatment significantly increased cleaved caspases 3, 9 and PARP in a time dependent manner, while the gene expression analysis of apoptosis related genes revealed only an elevated expression of the BIRC5 gene in Kasumi-1 cells after 24h. When screening for phospho-proteins, a decrease (0.5-fold) in phosphorylation was identified for the proteins: Phospholipase C gamma 1 Y783 (PLCγ1), Proline-rich tyrosine kinase 2 Y402, Endothelial nitric oxide synthase S1177 and Signal transducer and activator of transcription 3 (STAT3 Y705) in both cell lines upon HF treatment, suggesting that these proteins are primary targets of HF.In addition, when treated with HF the levels of STAT3 S727 and STAT5a/b Y694/Y699 were decreased in Kasumi-1, whereas the level of TP53 S392/S46/S15 was diminished in THP-1. In vivo, NSG mice transplanted with Kasumi-1 cells and treated with HF exhibited significantly longer OS (mean value: 144 days), compared to vehicle- treated controls (mean value: 94.5 days; P=.006).Conclusion: Our results demonstrated that HF exerts antileukemia activity, reducing the quantity of S-phase cells in sensitive cell lines and exhibiting a potent proapoptotic effect. The phosphoarray identified PLCγ1 and STAT 3/5 as targets of HF, corroborating the initiation of apoptosis upon treatment. In agreement with in vitro data, we demonstrated that NSG mice treated with HF presented longer OS, which provides new insights for the development of alternative/complementary therapeutic strategies for AML patients presenting a mutation in TP53 . DisclosuresNo relevant conflicts of interest to declare.

Anti-gastric cancer activity and mechanism of natural compound "Heilaohulignan C" isolated from Kadsura coccinea.

In this research, we analyzed the antitumor activity of one new compound Heilaohulignan C (B-6) on the human gastric carcinoma cells. MTT, cell migration, Calcein AM/Propidium Iodide (PI), and flow cytometry in BGC-823 cell line (gastric tumor). Western blot was utilized to distinguish the protein level. Xenografts nude mice were used for in vivo anticancer analysis. H&E staining and laboratory investigation was accomplished for toxicity study. MTT test demonstrated the cytotoxicity of BGC-823 cells, Calcein AM/Propidium Iodide (PI) examine indicated increment dead cells proportion with a high dose of B-6, Flow cytometry (FACS) measure showed that B-6 influenced gastric cancer cells by initiating apoptosis. Western blot analysis confirmed that (B-6) decrease the level of Bcl-2 and increase the level of p53, Bax, and cleaved Caspase-3, this confirms that the B-6 doing the apoptosis through caspase and cytochrome C apoptotic pathways. Also, B-6 particularly decline the tumor volume and tumor size in the xenograft mice. H&E staining additionally supports that B-6 does not have any toxic impact on the normal tissues. This research supports that B-6 have pharmacological activity against gastric cancer, by p53 and mitochondrial dependent apoptotic pathway, and have no toxicity on normal tissues.

Interleukin-6 sensitizes TNF-α and TRAIL/Apo2L dependent cell death through upregulation of death receptors in human cancer cells

Interleukin-6 (IL-6) enhanced TNF-α and TRAIL/Apo2L induced cell death in various human cancer cells derived from malignant glioma, melanoma, breast cancer and leukemia, although the effect was not detected with IL-6 alone. The effects of IL-6 using SKBR3 cells were associated with the generation of apoptotic cells as analyzed by fluorescence microscopy and flow cytometry. IL-6 activated p53 and upregulated TRAIL death receptors (DR-4 and DR-5) and stimulated the TNF-α and TRAIL dependent extrinsic apoptotic pathway without activation of the p53 mediated intrinsic apoptotic pathway. TNF-α and TRAIL induced cleavage of caspase-8 and caspase-3 was more enhanced by IL-6, although these caspases were not cleaved by IL-6 alone. The dead cell generation elicited by the combination with IL-6 was blocked by anti-human TRAIL R2/TNFRSF10B Fc chimera antibody which can neutralize the DR-5 mediated death signal. These findings indicate that IL-6 could contribute to the enhancement of TNF-α or TRAIL induced apoptosis through p53 dependent upregulation of DR-4 and DR-5. The data suggest that a favorable therapeutic interaction could occur between TNF-α or TRAIL and IL-6, and provide an experimental basis for rational clinical treatments in various cancers.

A Newly-Synthesized Chalcone Derivative of Ligustrazine Induces Caspase-Dependent and Apoptosis-Inducing Factor-Dependent Apoptosis in Cochlear Hair Cells

Objective: A newly synthesized derivative of ligustrazine chalcone, named as Z11, has shown a variety of promising biological activities. Here we aim to explore the effects of Z11 on the cochlear hair cells (HCs). Methods: Immunostaining and transmission electron microscopy (TEM) were used to examine the survival of HCs and their morphological changes. Furthermore, apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and the mRNA expression of apoptosis related genes including Caspase-9, Caspase-3, Bcl-2, Bax and Apaf1 were measured by RT-PCR. In addition, the protein expression of cleaved-Caspas-3 and cleaved-Caspase-9 were analyzed by Western blot respectively, and the protein expressionof AIF and cleaved-Caspase-3 were assessed by immunofluorescence as well. Results: Immunostaining showed that Z11 was ototoxic to mouse cochlear hair cells and significantly triggered cell death in a concentration-, time- and location-dependent manner. TUNEL assays evidenced that Z11 exerts its cytotoxicity through induction of apoptosis of cochlear hair cells in vitro. Immunofluorescence and western blot assay showed that Z11 activated the translation of apoptosis-inducing factor (AIF) and Caspase-9/Caspase-3 dependent apoptotic pathway in cochlear hair cells (HCs). Conclusion:These findings suggest that Z11 exhibits its ototoxicity through inducing apoptosis of HCs via both Caspase-dependent and AIF translocation pathways in mouse cochlear cultures.

Retinoic Acid Treatment of Human Hematological Malignancies Induces Caspase Dependent and Independent Apoptotic Cell Death

The unprejudiced of this education is to gauge the ability of the retinoic acid to induce apoptotic cell deathin hematological tumors through caspase dependent or independent apoptotic pathway, The cytotoxicityeffects of retinoic acid of different concentrations (400,350,300,250,200,150,100,50,25,12.5 µg\ml) andexposure for all hematological malignancy cell lines (Human non-Hodgkin lymphoma SR and humanmultiple myeloma (COLO 677) and Human Monocytic Leukemia THP1 and Acute promyelocytic leukemiaNB4) have been determined using a microtetrazolium (MTT) assay. Propodeum iodide and alcidine orange(AO/PI) paired discoloration was used to study the ability of retinoic acid to induce apoptosis in the infectedcells and examined under fluorescence microscope and quantified for the percentage of apoptosis induction.Quantitative immunocytochemistry assay was used to study the caspase dependent and independentproteins expression in infected and control cells. Cells treated with Retinoic Acid showed increased celldeath percentage compared to the untreated cells as quantified by MTT assay. AO/PI results revealed thatRetinoic Acid had powerful effect on inducing apoptosis significantly (p<0.001) in human cancer cell linestested, compared to control cell. Immunocytochemistry in Retinoic Acid infected human hematological celllines revealed remarkable increase in expression of caspase 8,9 (dependent pathway) and AIF, ENDOG(independent pathway) induces a significant (p<0.002) as compared untreated cell.This study, which shows the role of the Retinoic Acid in inducing apoptosis through a dependent andindependent pathway in cancer cells, we anticipation these annotations will shanty light on the impendingexploration of retinoic acid in cancer hindrance and rehabilitation

Micheliolide suppresses the viability, migration and invasion of U251MG cells via the NF-κB signaling pathway.

Micheliolide (MCL), a sesquiterpene lactone isolated from Michelia compressa and Michelia champaca, has been used previously to inhibit the NF-κB signaling pathway. MCL has exerted various therapeutic effects in numerous types of disease, such as inflammatory and cancer. However, to the best of our knowledge, its underlying anticancer mechanism remains to be understood. The present study aimed to investigate the effects of MCL on human glioma U251MG cells and to determine the potential anticancer mechanism of action of MCL. From Cell Counting Kit-8, colony formation assay, apoptosis assay and Confocal immunofluorescence imaging analysis, the results revealed that MCL significantly inhibited cell viability in vitro and induced cell apoptosis via activation of the cytochrome c/caspase-dependent apoptotic pathway. In addition, MCL also suppressed cell invasion and metastasis via the wound healing and Transwell invasion assays. Furthermore, western blot and reverse transcription PCR analyses demonstrated that MCL significantly downregulated cyclooxygenase-2 (COX-2) expression levels, which may have partially occurred through the inactivation of the NF-κB signaling pathway. In conclusion, the results of the present study indicated that MCL may inhibit glioma carcinoma growth by downregulating the NF-κB/COX-2 signaling pathway, which suggested that MCL may be a novel and alternative antitumor agent for the treatment of human glioma carcinoma.

A Chimeric Signal Peptide-Galectin-3 Conjugate Induces Glycosylation-Dependent Cancer Cell-Specific Apoptosis.

Exploitation of altered glycosylation in cancer is a major goal for the design of new cancer therapy. Here, we designed a novel secreted chimeric signal peptide-Galectin-3 conjugate (sGal-3) and investigated its ability to induce cancer-specific cell death by targeting aberrantly N-glycosylated cell surface receptors on cancer cells. sGal-3 was genetically engineered from Gal-3 by extending its N-terminus with a noncleavable signal peptide from tissue plasminogen activator. sGal-3 killing ability was tested on normal and tumor cells in vitro and its antitumor activity was evaluated in subcutaneous lung cancer and orthotopic malignant glioma models. The mechanism of killing was investigated through assays detecting sGal-3 interaction with specific glycans on the surface of tumor cells and the elicited downstream proapoptotic signaling. We found sGal-3 preferentially binds to β1 integrin on the surface of tumor cells due to aberrant N-glycosylation resulting from cancer-associated upregulation of several glycosyltransferases. This interaction induces potent cancer-specific death by triggering an oncoglycan-β1/calpain/caspase-9 proapoptotic signaling cascade. sGal-3 could reduce the growth of subcutaneous lung cancers and malignant gliomas in brain, leading to increased animal survival. We demonstrate that sGal-3 kills aberrantly glycosylated tumor cells and antagonizes tumor growth through a novel integrin β1-dependent cell-extrinsic apoptotic pathway. These findings provide proof-of-principle that aberrant N-oncoglycans represent valid cancer targets and support further translation of the chimeric sGal-3 peptide conjugate for cancer therapy.

Deglucohellebrin: A Potent Agent for Glioblastoma Treatment.

Glioblastoma is the most common primary brain tumor in adults with a dismal prognosis. To date, several anticancer agents have been isolated from plants. Helleborus odorus subsp. Cyclophyllus is an endemic plant of the Balcan flora. Herewith, we investigated for the first time, the anti-glioma effect of deglucohellebrin (DGH) extracted from the roots of Helleborus. We investigated the effect of DGH in U251MG, T98G and U87G glioblastoma cell lines. We selected the T98G cells because of their inherent temozolomide resistance. The IC50 value of reduced viability for DGH was 7x10-5M in U251MG cells, 5x10-5M for the T98G cells and 4x10-5M in U87G cells during 72h treatment. DGH induced G2/M cell cycle arrest, caspace-8 activation and significant mitochondrial membrane depolarization, suggesting the activation of the intrinsic, mitochondrial- dependent apoptotic pathway. DGH and temozolomide induced changes in CDs' expression in U251MG and T98G cells. In zebrafish, DGH did not induce toxicity or behavioral alterations. The present study is the first to determine the anti-glioma activity of DGH. DGH may be a potent agent for glioblastoma treatment and further studies are needed.