Fluorescein Isothiocyanate-annexin Research Articles

A High Dose of Calcitriol Inhibits Glycolysis and M2 Macrophage Polarization in the Tumor Microenvironment by Repressing mTOR Activation: in vitro and Molecular Docking Studies.

Macrophages interact with tumor cells within the tumor microenvironment (TME), which plays a crucial role in tumor progression. Cancer cells also can instruct macrophages to facilitate the spread of cancer and the growth of tumors. Thus, modulating macrophages-cancer cells interaction in the TME may be therapeutically beneficial. Although calcitriol (an active form of vitamin D) has anticancer properties, its role in TME is unclear. This study examined the role of calcitriol in the regulation of macrophages and cancer cells in the TME and its influence on the proliferation of breast cancer cells. We modeled the TME, in vitro, by collecting conditioned medium from cancer cells (CCM) and macrophages (MCM) and culturing each cell type separately with and without (control) a high-dose (0.5 µM) calcitriol (an active form of vitamin D). An MTT assay was used to examine cell viability. Apoptosis was detected using FITC (fluorescein isothiocyanate) annexin V apoptosis detection kit. Western blotting was used to separate and identify proteins. Quantitative real-time PCR was used to analyze gene expression. Molecular docking studies were performed to evaluate the binding type and interactions of calcitriol to the GLUT1 and mTORC1 ligand-binding sites. Calcitriol treatment suppressed the expression of genes and proteins implicated in glycolysis (GLUT1, HKII, LDHA), promoted cancer cell apoptosis, and reduced viability and Cyclin D1gene expression in MCM-induced breast cancer cells. Additionally, calcitriol treatment suppressed mTOR activation in MCM-induced breast cancer cells. Molecular docking studies further showed efficient binding of calcitriol with GLUT1 and mTORC1. Calcitriol also inhibited CCM-mediated induction of CD206 and increased TNFα gene expression in THP1-derived macrophages. The results suggest that calcitriol may impact breast cancer progression by inhibiting glycolysis and M2 macrophage polarization via regulating mTOR activation in the TME and warrants further investigation in vivo.

Induction of apoptosis by essential oil of Dracocephalum kotschyi on Trichomonas vaginalis.

Trichomonas vaginalis is a protist parasite that causes trichomoniasis, a sexually transmitted disease. Metronidazole is the current treatment for trichomoniasis. However, this drug can provoke severe side effects, and some strains present resistance, making the development of alternative treatments for trichomoniasis urgent. We investigate the use of essential oil obtained from Dracocephalum kotschyi on T. vaginalis. D. kotschyi has antispasmodic and analgesic properties and is well known in Iran. The essential oil was obtained by hydrodistillation from 1000 g of the powdered plant. Gas chromatography-mass spectrometry analysis was used for the chemical composition of the essential oil, and 11 substances were identified, corresponding to 91.5% of the oil. Copaene (22.15%), Methyl geranate (16.31%), Geranial (13.78%) and Carvone (11.34%) were the main substances. A cell viability test was used to determine the percentage of growth inhibition (GI%) and the half-maximal inhibitory concentration (IC50) on T. vaginalis after incubation with the prepared essential oil. The oil induced an IC50 of 84.07 µg/ml after 24 h contact with trophozoites. Cytotoxicity was determined by MTT assay on the J774.A1 haematopoietic cell line. In addition, the initial stage of apoptosis was assayed using the fluorescein isothiocyanate Annexin V Apoptosis Detection Kit. Evaluation of the in vitro anti-trichomonal properties of D. kotschyi essential oils showed that it effectively induces apoptosis on T. vaginalis between 100 and 700 µg/ml after 48 h without toxicity on haematopoietic cells, suggesting that D. kotschyi essential oil can induce programmed death in T. vaginalis. The anti-trichomonal properties of D. kotschyi essential oil indicate that they could be suitable for new pharmacologic studies after new tests with human vaginal epithelial cells.

The Cytotoxicity and Genotoxicity of Bioactive Dental Materials.

The promotion of biologically based treatment strategies in restorative dentistry is of paramount importance, as invasive treatments should be avoided to maintain the tooth’s vitality. This study aimed to assess the biocompatibility of commercially available bioactive materials that can be used for dental pulp capping. The study was performed with a monocyte/macrophage peripheral blood SC cell line (ATCC CRL-9855) on the following six specific bioactive materials: ProRoot MTA (Dentsply Sirona), MTA Angelus (Angelus), Biodentine (Septodont), TheraCal LC (Bisco), ACTIVA BioACTIVE (Pulpdent) and Predicta Bioactive Bulk (Parkell). The cytotoxicity of the investigated agents was measured using a resazurin-based cell viability assay, while the genotoxicity was evaluated using an alkaline comet assay. Additionally, flow cytometry (FC) apoptosis detection was conducted with a FITC (fluorescein isothiocyanate) Annexin V Apoptosis Detection Kit I. FC cell-cycle arrest assessment was carried out with propidium iodide staining. The results of this study showed no significant cytotoxicity and genotoxicity (p > 0.05) in ProRoot MTA, MTA Angelus, Biodentine, ACTIVA BioACTIVE and Predicta Bioactive. Conversely, TheraCal LC presented a significant decrease (p < 0.001). In conclusion, due to excellent biocompatibility and low cytotoxicity, MTA, Biodentine, ACTIVA BioACTIVE and Predicta Bioactive may be suitable for pulp capping treatments. On the other hand, due to the high cytotoxicity of TheraCal LC, its use should be avoided in vital pulp therapies.

Parthenolide reverses the epithelial to mesenchymal transition process in breast cancer by targeting TGFbeta1: In vitro and in silico studies

AimsBreast cancer metastasis is the leading cause of mortality among breast cancer patients. Epithelial to mesenchymal transition (EMT) is a biological process that plays a fundamental role in facilitating breast cancer metastasis. The present study assessed the efficacy of parthenolide (PTL Tanacetum parthenium) on EMT and its underlying mechanisms in both lowly metastatic, estrogen-receptor positive, MCF-7 cells and highly metastatic, triple-negative MDA-MB-231 cells. Main methodsMCF-7 and MDA-MB-231 cells were treated with PTL (2 μM and 5 μM). Cell viability was determined by MTT (3-(4,5-dimethy lthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Apoptosis was analyzed by the FITC (fluorescein isothiocyanate) annexin V apoptosis detection kit. The monolayer wound scratch assay was employed to evaluate cancer cell migration. Proteins were separated and identified by Western blotting. Gene expression was analyzed by quantitative real-time PCR. Key findingsPTL treatment significantly reduced cell viability and migration while inducing apoptosis in both cell lines. Also, PTL treatment reverses the EMT process by decreasing the mesenchymal marker vimentin and increasing the epithelial marker E-cadherin compared to the control treatment. Importantly, PTL downregulates TWIST1 (a transcription factor and regulator of EMT) gene expression, concomitant with the reduction of transforming growth factor beta1 (TGFβ1) protein and gene expression in both cell lines. Additionally, molecular docking studies suggest that PTL may induce anticancer properties by targeting TGFβ1 in both breast cancer cell lines. SignificanceOur findings provide insights into the therapeutic potential of PTL to mitigate EMT and breast cancer metastasis. These promising results demand in vivo studies.

Inhibiting nonhomologous end-joining repair would promote the antitumor activity of gemcitabine in nonsmall cell lung cancer cell lines.

Nonsmall cell lung cancer (NSCLC) is a major type of lung cancer. In current study, we aim to evaluate whether the combination of Ku70/80 heterodimer protein inhibitor STL127705 and gemcitabine would be more favorable approach for the treatment of NSCLC compared with monotreatment with gemcitabine. Clongenic survival assay was used to determine the survival and sensitivity to irradiation. H1299 was stained with fluorescein isothiocyanate-Annexin V, and cell apoptosis was measured by flow cytometry. H1299 cells were transfected with nonhomologous end-joining (NHEJ) repair reporter, and stable cell line was selected by puromycin. NHEJ activity was determined based on the intensity of green fluorescent protein. DNA double-strand breaks (DSBs) were determined by the fluorescence intensity of γH2AX using flow cytometry. The mRNA expressions of Ku70 and Ku80 were determined using quantitative real-time PCR. Combination of STL127705 enhanced sensitivity of NSCLC cell lines to irradiation when compared with treatment with gemcitabine alone. However, small cell lung cancer cell line was not affected. H1299 cells treated with STL127705 in combination with gemcitabine showed a significantly increased apoptosis compared with H1299 cells treated with gemcitabine alone. Moreover, STL127705 treatment dramatically reduced NHEJ activity in H1299 cells when compared with gemcitabine single treatment. Increased DSBs were consistently observed in H1299 when treated with the combination of STL127705 and gemcitabine. However, the mRNA levels of Ku70 and Ku80 were upregulated by the combination treatment. It demonstrated that STL127705 enhanced antitumor activity of gemcitabine. Mechanistically, treatment with STL127705 enhanced DNA damage via inhibiting NHEJ pathway, blocking DNA-PK, and forming Ku70/80 heterodimer, eventually leading to tumor cells apoptosis.

The RNA-Binding Protein DDX18 Promotes Gastric Cancer by Affecting the Maturation of MicroRNA-21.

ObjectivesThe noncoding RNAs (ncRNAs) play important roles in gastric cancer. Most studies have focused on the functions and influence of ncRNAs, but seldom on their maturation. DEAD box genes are a family of RNA-binding proteins that may influence the development of ncRNAs, which attracted our attention. By combining a small sample for high-throughput gene microarray screening with large samples of The Cancer Genome Atlas (TCGA) data and our cohort, we aimed to find some gastric cancer-related genes. We evaluated the clinical significance and prognostic value of candidate gene DDX18, which is overexpressed in gastric cancer tissues. To provide a theoretical basis for the development of new therapeutic targets for the treatment of gastric cancer, we investigated its effect on the malignant biological behavior of gastric cancer in vitro and in vivo, and also discuss its mechanism of action.Methods(i) The differential profiling of mRNA expression in five pairs of gastric cancer and adjacent normal tissues was studied by Arraystar Human mRNA Microarray. By combining this with TCGA data and our cohort, we finally filtered out DDX18, which was upregulated in gastric cancer tissues, for further investigation. (ii) The protein expression of DDX18 was detected by immunohistochemistry staining. Then the relationship between the DDX18 expression level and the clinicopathological data and prognosis was analyzed. (iii) A CCK-8 assay and colony formation assay were used to evaluate the effect of DDX18 on cell growth and proliferation in vitro. A transwell assay was also performed to examine the migration and invasion of gastric cancer cells. Cell apoptosis was analyzed by using a fluorescein isothiocyanate–annexin V/propidium iodide double-staining assay. To identify the role of DDX18 in the tumorigenic ability of gastric cancer cells in vivo, we also established a subcutaneous gastric cancer xenograft model. Coimmunoprecipitation, small RNAseq, and western blotting were performed to explore the mechanism of action of DDX18 in gastric cancer. A patient-derived xenograft (PDX) model was used to confirm the effect of DDX18 in gastric cancer tissues.Result(i) DDX18 was upregulated in gastric cancer tumor tissues from a TCGA database and our cohort. The expression of DDX18 was also closely related to tumor volume, Borrmann classification, degree of tumor differentiation, cancer embolus, lymph node metastasis, and TNM stage. (ii) DDX18 could promote cell proliferation, migration, and invasion and inhibit cell apoptosis in vivo and in vitro. (iii) DDX18 could promote the maturation of microRNA-21 through direct interaction with Drosha, decreasing PTEN, which could upregulate the AKT signaling pathway. (iv) The PDX model showed that DDX18 could promote the proliferation of gastric cancer tissues by means of the PTEN–AKT signaling pathway.Conclusions(i) DDX18 can be treated as a molecular marker to assess the prognosis of patients with gastric cancer. (ii) DDX18 could be a potential therapeutic target in gastric cancer.

The apoptotic and free radical–scavenging abilities of the protein hydrolysate obtained from chicken feather meal

This study examined the antioxidant capabilities of peptides derived from chicken feather meal (CFM) protein hydrolysates which were produced using 3 different microbial proteases (Neutrase, Alcalase, and flavourzyme) and tested at varying concentrations, namely 1, 2, and 5% by weight. The highest levels of 2,2-diphenyl-1-picrylhydrazl (DPPH) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical scavenging activities were presented by CFM hydrolysate derived using 5 wt% Neutrase and digested for 4 h. Fractionation of this particular hydrolysate was then performed by applying 10, 5, 3, and 0.65 kDa molecular weight cutoff membranes. It was then determined that the molecular weight (MW) < 0.65 kDa fraction achieved the greatest level of free radical scavenging activity in the context of DPPH and ABTS. The MW < 0.65 kDa fraction then underwent additional fractionation using reverse-phase high-performance liquid chromatography to derive 3 main fractions designated as F1, F2, and F3. All of these fractions presented a high level of activity in DPPH radical scavenging, although no significant ABTS scavenging was observed. Quadrupole time-of-flight tandem mass spectrometry was used in determining the peptide contents of the fractions as Phe-Asp-Asp-Arg-Gly-Arg-X for F1 (FDDRGRX, 875 Da), Val-Thr-Leu-Ala-Val-Thr-Lys-His for F2 (VTLAVTKH, 868 Da), and Val-Ser-Glu-Ile-X-Ser-Ile-Pro-Ile-Ser for F3 (VSEIXSIPIS, 1,055 Da). Moreover, the F2 fraction was shown to be capable of preventing DNA damage induced by hydroxyl radicals, as indicated in tests using the plasmids pKS, pUC19, and pBR322 via the Fenton reaction. This outcome was demonstrated through in vitro antiproliferative activity in human cell lines based on SW620 colon cancer, using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The F2 fraction at 0.5 wt.% was also shown to be capable of inducing weak early apoptosis, which could be measured by using the Fluorescein isothiocyanate Annexin V Apoptosis Detection Kit with Propidium Iodide Solution. Furthermore, an increase in caspase-3 and caspase-8 activity was observed in SW620 cells following exposure for 24 h and 48 h.

Repression of Death Receptor–Mediated Apoptosis of Hepatocytes by Hepatitis B Virus e Antigen

Hepatitis B virus (HBV) e antigen (HBeAg) is associated with viral persistence and pathogenesis. Resistance of HBV-infected hepatocytes to apoptosis is seen as one of the primary promotors for HBV chronicity and malignancy. Fas receptor/ligand (Fas/FasL) and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) system plays a key role in hepatic death during HBV infection. We found that HBeAg mediates resistance of hepatocytes to FasL or TRAIL-induced apoptosis. Introduction of HBeAg into human hepatocytes rendered resistance to FasL or TRAIL cytotoxicity in a p53-dependent manner. HBeAg further inhibited the expression of p53, total Fas, membrane-bound Fas, TNF receptor superfamily member 10a, and TNF receptor superfamily member 10b at both mRNA and protein levels. Incontrast, HBeAg enhanced the expression of soluble forms of Fas through facilitation of Fas alternative mRNA splicing. In a mouse model, expression of HBeAg in mice injected with recombinant adenovirus-associated virus 8 inhibited agonistic anti-Fas antibody-induced hepatic apoptosis. Xenograft tumorigenicity assay also found that HBeAg-induced carcinogenesis was resistant to the proapoptotic effect of TRAIL and chemotherapeutic drugs. These results indicate that HBeAg may prevent hepatocytes from FasL and TRAIL-induced apoptosis by regulating the expression of the proapoptotic and antiapoptotic forms of death receptors, which may contribute to the survival and persistence of infected hepatocytes during HBV infection.

CKD-602, a topoisomerase I inhibitor, induces apoptosis and cell-cycle arrest and inhibits invasion in cervical cancer

BackgroundCervical cancer is the third most common gynecological malignancy. Conventional treatment options are known to be ineffective for the majority of patients with advanced or recurrent cervical cancer. Therefore, novel therapeutic agents for cervical cancer are necessary. In this study, the effects of CKD-602 in cervical cancer were investigated.MethodsThree established human, immortalized, cervical cancer cell lines (CaSki, HeLa and SiHa) were used in this study. Following treatment with CKD-602, apoptosis was quantified using fluorescein isothiocyanate Annexin V-FITC and propidium iodide (PI) detection kit and cell cycle analysis was analyzed using fluorescence activated cell sorting (FACS). Transwell chambers were used for invasion assays. Western blot assay was performed to analyze proteomics. CaSki cells were subcutaneously injected into BALB/c-nude mice and cervical cancer xenograft model was established to elucidate the antitumor effect of CKD-602 in vivo.ResultsTreatment with CKD-602 induced apoptosis and increased expression of the enzyme PARP, cleaved PARP, and BAX. In addition, expression of phosphorylated p53 increased. Cell cycle arrest at G2/M phase and inhibition of invasion were detected after treatment with CKD-602. A significant decrease in cervical cancer tumor volume was observed in this in vivo model, following treatment with CKD-602.ConclusionsThis is the first report of CKD-602 having an antitumor effect in cervical cancer in both an in vitro and in vivo models. The results of this study indicate that CKD-602 may be a novel potential drug, targeting cervical cancer, providing new opportunities in the development of new therapeutic strategies.

α-hederin induces autophagic cell death in colorectal cancer cells through reactive oxygen species dependent AMPK/mTOR signaling pathway activation.

α-hederin, a monodesmosidic triterpenoid saponin, had previously demonstrated strong anticancer effects. In the current study, the pharmacological mechanism of autophagic cell death induced by α-hederin was investigated in human colorectal cancer cells. First, through cell counting kit-8 and colony formation assays, it was demonstrated that α-hederin could inhibit the proliferation of HCT116 and HCT8 cell. Results of flow cytometry using fluorescein isothiocyanate Annexin V/propidium iodide and Hoechst 33258 staining indicated that α-hederin could induce apoptosis. Western blotting demonstrated that α-hederin could activate mitochondrial apoptosis signal pathway. Then, using light chain 3 lentiviral and electron microscope assay, it was demonstrated that α-hederin could induce autophagy in colorectal cancer cells. In addition, immunohistochemistry results from in vivo experiments also demonstrated that α-hederin could induce autophagy. AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling was demonstrated to be activated by α-hederin, which could be blocked by reactive oxygen species (ROS) inhibitor NAC. Furthermore, NAC could inhibit apoptosis and autophagy induced by α-hederin. Finally, 3-MA (autophagy inhibitor) reduced the inhibition of α-hederin on cell activity, but it had no significant effect on apoptosis. In conclusion, α-hederin triggered apoptosis through ROS-activated mitochondrial signaling pathway and autophagic cell death through ROS dependent AMPK/mTOR signaling pathway activation in colorectal cancer cells.

Inhibition of XIAP increases carboplatin sensitivity in ovarian cancer

PurposeCarboplatin is a first-line treatment for ovarian cancer. However, most patients develop resistance and undergo disease recurrence. This study aims to explore the relationship between the expression of X-linked inhibitor of apoptosis protein (XIAP) and carboplatin sensitivity in ovarian cancer.Patients and methodsWe examined the expression of XIAP in ovarian cancer by immuno-chemistry. Next, we investigated the role of XIAP in regulating carboplatin sensitivity in ovarian cancer ES2 and 3AO cells through Cell Counting Kit-8 cell viability assay and fluorescein isothiocyanate-Annexin V/propidium iodide apoptosis assay. Expression of apoptotic effectors was measured by Western blot.ResultsThe immunochemistry results showed that high XIAP expression levels inversely correlated with carboplatin response (P=0.03) and progression-free survival (P=0.0068) in patients with ovarian cancer. Knockdown of XIAP repressed the cell viabilities in the carboplatin-treated cells and increased carboplatin-induced caspase activation. In summary, our data show that XIAP mediates carboplatin sensitivity of ovarian cancer.ConclusionIn summary, our data show that XIAP mediates carboplatin sensitivity of ovarian cancer and XIAP may be a novel target for the treatment of carboplatin-resistant ovarian cancer.

Overexpression of Wilms' tumor 1 in skin lesions of psoriasis is associated with abnormal proliferation and apoptosis of keratinocytes.

Psoriasis vulgaris (PV) is a chronic inflammatory skin disease, which is characterized by the abnormal proliferation and apoptosis of keratinocytes. Previous studies have demonstrated that transcription factor Wilms' tumor 1 (WT1) is involved in a number of pathophysiological processes, including organ development, tumorigenesis and cell proliferation. However, the role of WT1 in PV remains unclear. In the present study, WT1 expression was analyzed by reverse transcription‑quantitative polymerase chain reaction and western blot analyses. WT1 was stably overexpressed or inhibited in HaCaT cells using Lipofectamine® 2000, and cell proliferation and apoptosis were determined using a Cell Counting Kit‑8 or Fluorescein Isothiocyanate Annexin V Apoptosis Detection kit II, respectively. We demonstrated that compared with normal controls, the mRNA and protein expression levels of WT1 were significantly increased in non‑lesional skins (human, P<0.0001 and P=0.0291, respectively; mouse, P=0.0020 and P=0.0150, respectively) and lesional skins (human, P<0.0001 and P=0.0060, respectively; mouse, P=0.0010 and P=0.0172, respectively) of patients with PV, in addition to the imiquimod (IMQ)‑induced psoriasis‑like mouse model. WT1 mRNA and protein expression levels in lesional skins were slightly increased compared with those in non‑lesional skins from patients with psoriasis (P=0.2510 and P=0.1690, respectively) and IMQ‑treated mice (P=0.9590 and P=0.2552, respectively), although there were no statistical differences. Knockdown of WT1 inhibited the proliferation of HaCaT cells [day (D)4, P=0.0454; D5, P=0.0021] and promoted their apoptosis (P=0.0007), while overexpressing WT1 exhibited the opposite effects (proliferation D3, P=0.0216; D4, P=0.0356; D5, P=0.0188; apoptosis, P=0.0003). Furthermore, it was identified that the inflammatory cytokines interleukin‑17A (IL‑17A), interferon‑γ and IL‑22 induced the overexpression of WT1 in HaCaT cells. The results of the present study suggested that inflammatory cytokine‑induced WT1 overexpression may promote the formation of psoriatic skin lesions via regulation of the proliferation and apoptosis of keratinocytes.

Vitamins C and K3: A Powerful Redox System for Sensitizing Leukemia Lymphocytes to Everolimus and Barasertib

Recent studies provided convincing evidence for the anticancer activity of combined application of vitamin C and pro-vitamin K3 (menadione). The molecular pathways underlying this process are still not well established. The present study aimed to investigate the effect of the combination of vitamin C plus pro-vitamin K3 on the redox status of leukemia and normal lymphocytes, as well as their sensitizing effect for a variety of anticancer drugs. Cytotoxicity of the substances was analyzed by trypan blue staining and automated counting of live and dead cells. Apoptosis was analyzed by fluorescein isothiocyanate-annexin V test. Oxidative stress was evaluated by the intracellular levels of reactive oxygen and nitrogen species and protein-carbonyl products. Combined administration of 300 μM vitamin C plus 3 μM pro-vitamin K3 reduced the viability of leukemia lymphocytes by ~20%, but did not influence the viability of normal lymphocytes. All combinations of anticancer drug plus vitamins C and K3 were characterized by synergistic cytotoxicity towards Jurkat cells, compared to cells treated with drug alone for 24 h. In the case of barasertib and everolimus, this synergistic cytotoxicity increased within 72 hours. It was accompanied by strong induction of apoptosis, but a reduction of level of hydroperoxides and moderately increased protein-carbonyl products in leukemia cells. Leukemia lymphocytes were more sensitive to combined administration of anticancer drug (everolimus or barasertib) plus vitamins C and K3, compared to normal lymphocytes. The combination of vitamin C plus K3 seems to be a powerful redox system that could specifically influence redox homeostasis of leukemia cells and sensitize them to conventional chemotherapy.

Adhesion, proliferation, and apoptosis in different molecular portraits of breast cancer treated with silver nanoparticles and its pathway-network analysis.

BackgroundSilver nanoparticles (AgNPs) have attracted considerable attention due to the variety of their applications in medicine and other sciences. AgNPs have been used in vitro for treatment of various diseases, such as hepatitis B and herpes simplex infections as well as colon, cervical, and lung cancers. In this study, we assessed the effect on proliferation, adhesion, and apoptosis in breast cancer cell lines of different molecular profiles (MCF7, HCC1954, and HCC70) exposed to AgNPs (2–9 nm).MethodsBreast cancer cell lines were incubated in vitro; MTT assay was used to assess proliferation. Adhesion was determined by real-time analysis with the xCELLingence system. Propidium iodide and fluorescein isothiocyanate-Annexin V assay were used to measure apoptosis. The transcriptome was assessed by gene expression microarray and Probabilistic Graphical Model (PGM) analyses.ResultsThe results showed a decreased adhesion in breast cancer cell lines and the control exposed to AgNPs was noted in 24 hours (p≤0.05). We observed a significant reduction in the proliferation of MCF7 and HCC70, but not in HCC1954. Apoptotic activity was seen in all cell lines exposed to AgNPs, with an apoptosis percentage of more than 60% in cancer cell lines and less than 60% in the control. PGM analysis confirmed, to some extent, the effects of AgNPs primarily on adhesion by changes in the extracellular matrix.ConclusionExposure to AgNPs causes an antiproliferative, apoptotic, and anti-adhesive effect in breast cancer cell lines cultured in vitro. More research is needed to evaluate the potential use of AgNPs to treat different molecular profiles of breast cancer in humans.

Inhibition of autophagy attenuated curcumol-induced apoptosis in MG-63 human osteosarcoma cells via Janus kinase signaling pathway.

The present study aimed to investigate whether autophagy was triggered by curcumol and to explore the association between autophagy and apoptosis of MG-63 cells and the underlying mechanism. MG-63 cells were cultured in vitro. An MTT assay was performed to evaluate the proliferation inhibition of the MG-63 osteosarcoma cell line by curcumol. Fluorescein isothiocyanate-Annexin V/propidium iodide staining flow cytometry was performed to analyze the apoptotic rate of cells. The morphological alterations of cell nuclei were evaluated by Hoechst 33258 viable cell staining. The effects of autophagy in cells was investigated by green fluorescent protein (GFP)-light chain 3 (LC3) transfection and using a fluorescence microscope. The expression levels of LC3II, LC3I and cleaved caspase-3 and Janus kinase (JNK) signaling pathway activation were determined by western blot analysis. Cell proliferation was inhibited by curcumol in a dose- and time-dependent manner. Curcumol induced apoptosis by the caspase-dependent signaling pathway in MG-63 cells. The present study demonstrated that curcumol could induce autophagy of MG-63 cells, which was evaluated by transmission electron microscopy. Compared with the curcumol treatment alone group, the GFP-LC3-transfected green fluorescence plasmids and the LC3II/LC3I levels in cells of the curcumol and chloroquine (CQ) treatment group were upregulated, and the apoptotic ratio was downregulated following pretreatment with autophagy inhibitor CQ for 1 h. Furthermore, curcumol treatment induced phosphorylation of the JNK signaling pathway. Of note, pretreatment with the JNK inhibitor, SP600125, decreased the rates of autophagy and apoptosis, suggesting a crucial role served by the JNK signaling pathway in the activation of autophagy by curcumol. Taken together, the results of the present study suggested that activation of the JNK signaling pathway was involved in curcumol-induced autophagy. Curcumol is a novel drug for chemotherapeutic combination therapy. Curcumol demonstrated potential antitumor activities in MG-63 cells and may be used as a novel effective reagent in the treatment of osteosarcoma.

Protective mechanisms of hypaconitine and glycyrrhetinic acid compatibility in oxygen and glucose deprivation injury.

This study investigated the protective effect of the compatibility of hypaconitine (HA) and glycyrrhetinic acid (GA) on H9c2 cells under oxygen and glucose deprivation (OGD)-induced injury, and the possible mechanisms. We found that HA+GA significantly improved pathology and morphology of the nucleus and ultrastructure of H9c2 cells under OGD as determined by Hoechst 33342 staining and transmission electron microscopy (TEM) tests. It also reduced the releases of lactate dehydrogenase (LDH), creatine kinase-myocardial band isoenzyme (CK-MB), and aspartate transaminase (AST) from the cultured supernatant of H9c2 cells, which were tested by enzyme-linked immune sorbent assay (ELISA) kits. In addition, it lessened the apoptotic rate as determined by a fluorescein isothiocyanate-annexin V/propidium iodide (FITC-AV/PI) double staining assay. It was also found that HA+GA might regulate the protein expression associated with the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Overall, the study demonstrated that HA+GA protected H9c2 cells against OGD-induced injury, and the signaling mechanism might be related to the PI3K/Akt signaling pathway.

Anticancer Properties of Phyto-Synthesized Silver Nanoparticles from Medicinal Plant Artemisia tschernieviana Besser Aerial Parts Extract Toward HT29 Human Colon Adenocarcinoma Cells

This study highlights the apoptosis-inducing ability of eco-friendly phyto-synthesis route for silver nanoparticles (AgNPs) by using Artemisia tschernieviana extract (ATE). The aerial parts extract of A. tschernieviana was analysed for chemical composition using the gas chromatography–mass spectrometer (GC–MS) analysis. The phyto-synthesized AgNPs using A. tschernieviana extract (AgNP-ATE) were characterized by ultraviolet–visible, zeta potential, Fourier transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy. The AgNP-ATE thus prepared was observed to have characteristic surface plasmon resonance peak at around 425 nm. The surface morphology and size of AgNPs show that the particles were mostly spherical and their sizes varied from 5 to 50 nm. The prepared AgNP-ATE showed more potent in vitro cytotoxicity against HT29 human colon adenocarcinoma cell line than normal human embryonic kidney (HEK293) cells. Moreover, studies such as Fluorescein isothiocyanate-annexin V and propidium iodide staining, caspase assay, and Hoecht 33258 staining demonstrate that the prepared AgNP-ATE shows significantly enhanced apoptosis in human colon cancer cells as compared to untreated cells. The current data suggest that the phyto-synthesized AgNPs show effective anticancer properties against HT29 cells.

MiR-17-92 represses PTPROt and PP2A phosphatases and amplifies tonic BCR signaling in DLBCL cells.

B-cell receptor (BCR) signaling plays a pivotal role in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) and targeting the BCR pathway is a highly promising therapeutic strategy in this malignancy. The oncogenic microRNA miR-17-92 modulates multiple cellular processes such as survival, proliferation, apoptosis, angiogenesis, and BCR signaling. In the present study, we identified new targets of miR-17-92, PTPROt (protein phosphatase, receptor type O, truncated) and PP2A (protein phosphatase 2A) phosphatases, which regulate the activity of spleen tyrosine kinase (SYK) and AKT, critical components of BCR signal transduction in DLBCL cells. Introduction of miR-17-92 into DLBCL cells dampened the expression of the PTPROt and PP2A regulatory subunits PPP2R2A (protein phosphatase 2, regulatory subunit B, alpha) and PPP2R5E (protein phosphatase 2, regulatory subunit B, epsilon isoform) and increased the magnitude of SYK and AKT phosphorylation upon BCR ligation. Finally, we found that miR-17-92 expression modulates response to inhibitors of BCR signaling because downregulation of miR-17-92 increased SYK inhibitor-mediated toxicity in DLBCL cells. Our study reveals novel posttranscriptional regulatory pathways that contribute to the deregulation of BCR signaling and modulate SYK inhibitor activity in DLBCL.

Asiatic acid, a non-detergent type spermicide: exploration of plausible pathway of spermicidal action

The spermicidal and microbicidal activity of Asiatic acid isolated from Shorea robusta resin was explored earlier using rat spermatozoa. The current investigation is aimed at examining the spermicidal efficacy of the molecule against human spermatozoa along with the plausible pathway involved in its spermicidal action. Spermicidal efficacy of Asiatic acid against human spermatozoa was evaluated in vitro by modified Sander-Cramer test and the subsequent mode of spermicidal action was assessed by (a) hypo-osmotic swelling test, (b) double fluoroprobe staining, (c) flow cytometric detection of apoptosis by fluorescein isothiocyanate-Annexin V labelling and (d) JC-1 labelling. The minimum effective concentration of the molecule against human spermatozoa was found to be 500 µg/ml which is comparable to the standard spermicide nonoxynol-9 (minimum effective concentration 550 µg/ml). The mechanistic pathway of spermicidal action of the molecule involves apoptosis rather than general cell necrosis. The molecule Asiatic acid may be explored as an ingredient for the formulation of a new generation non-detergent type microbicidal contraceptive.

Z-venusol, from Gunnera perpensa, induces apoptotic cell death in breast cancer cells in vitro

Many African traditional medicines are used although their pharmacology is unclear. In this study, we investigated the in vitro anti-tumour effects of pure Z-venusol, a compound isolated from an indigenous South African plant called Gunnera perpensa. We also compared these to those of a commercially available concoction purported to contain crude extracts of G. perpensa.Pure Z-venusol, previously isolated from the roots of G. perpensa, was incubated with human breast (the MCF-7s) cancer cells and human mammary epithelial cells (HMECs). Proliferation was assessed using the sulforhodamine B (SRB) assays. The fluorescein isothiocyanate (FITC) Annexin V and the lactate dehydrogenase (LDH) activity assays were conducted to determine whether cell death, if any, was apoptotic or necrotic. The drugs used as positive controls included cisplatin and camptothecin. Re-Joovena™, a concoction claiming to contain G. perpensa (0.3mg/ml) and Ocotea bullata (0.3mg/ml), was also investigated.Z-venusol demonstrated a statistically significant, concentration-dependent, apoptotic inhibitory effect on proliferation of MCF-7 cells, with an IC50 of 53.7μg/ml (95% CI, 29.7–96.9.9μg/ml) after 72hr exposure, while the highest concentration (250μg/ml) used resulted in 69% inhibition. The FITC Annexin V and LDH results suggested that apoptosis contributed most of the effect observed. Further, there was insignificant inhibition (20%) of HMEC proliferation observed when the concentration of Z-venusol was increased beyond 16.6μg/ml. The highest concentration used resulted in only 27% inhibition of proliferation of HMEC cells. None of the Re-Joovena™ concentrations tested showed any significant effect. As was expected, cisplatin-induced inhibition of cell proliferation and camptothecin, at lower concentration (0.15µM), resulted in apoptotic cell death, and at high (≥4µM) concentrations, it induced necrotic cell death.Our results suggest that Z-venusol is cytotoxic to human breast tumour cells in vitro. While some cell death follows a necrotic pathway, the major mechanism of death appears to be apoptosis. The molecular mechanism of the observed effects is currently being investigated, in particular the relationship between Z-venusol and β-adrenergic receptors.