We investigated the molecular mechanism of apoptosis induced by novel jatropha-6(17),11E-diene class derivatives, compounds A, B, and C that were extracted from Euphorbia osyridea Boiss, in the ovarian cancer cell lines Caov-4 and OVCAR-3. The OVCAR-3 and Caov-4 cell lines were treated with different concentrations of these compounds. Cytotoxicity was evaluated using MTT, clonogenic survival assay, and flow cytometry assays. The production of reactive oxygen species (ROS), mitochondrial membrane potential (Δ), and the activity of caspase 3 and 9 were evaluated. Compounds A, B, and C reduced cell viability in a dose-dependent manner (P < 0.05). The IC values were calculated as 46.27 ± 3.86, and 38.81 ± 3.30 μmol/L for compound A, 36.48 ± 3.18 and 42.59 ± 4.50 μmol/L for compound B, and 85.86 ± 6.75 and 75.65 ± 2.56 μmol/L for compound C against the Caov-4 and OVCAR-3 cell lines, respectively. Apoptosis evaluation showed that jatrophane derivatives increase both early and late apoptosis (P < 0.01). These compounds also increased ROS generation, Δ, and the activity of caspase 3 and 9 in the treated cells. These results showed that compounds A and B have significant inhibitory effects on OVCAR-3 and Caov-4 proliferation and induction of apoptosis.

Natural products are considered recently as one of the source for production of efficient therapeutical agents for breast cancer treatment. In this study, a sesquiterpene lactone, 13‐O‐acetylsolstitialin A (13ASA), isolated from Centaurea cyanus, showed cytotoxic activities against MCF‐7 and MDA‐MB‐231 breast cancer cell lines using standard 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. To find the mechanism of action of cytotoxicity, annexin V/propidium iodide (PI) staining was performed for evaluation of apoptosis. This process was further confirmed by immunoblotting of anti‐ and proapoptotic, Bcl‐2 and Bax, proteins. Cell cycle arrest was evaluated by measurement of fluorescence intensity of PI dye and further confirmed by immunoblotting of Cdk‐4 and cyclin D1. Mitochondrial transmembrane potential (ΔΨm) and generation of reactive oxygen species (ROS) were measured using the JC‐1 and DCFDA fluorescence probes, respectively. These experiments showed that 13ASA is a potent cytotoxic agent, which activates apoptosis‐mediated cell death. In response to this compound, Bax/Bcl‐2 ratio was noticeably increased in MCF‐7 and MDA‐MB‐231 cells. Moreover, 13ASA induced cell cycle arrest at subG1 and G1 phases by decreasing protein levels of cyclin D1 and Cdk‐4. It was done possibly through the decrease of ΔΨm and increase of ROS levels which induce apoptosis. In conclusion, this study mentioned that 13ASA inhibit the growth of MCF‐7 and MDA‐MB‐231 breast cancer cell lines through the induction of cell cycle arrest, which triggers apoptotic pathways. 13ASA can be considered as a susceptible compound for further investigation in breast cancer study.

Endoplasmic reticulum (ER) stress is one of the main molecular events underlying pancreatic beta cell (PBC) failure, apoptosis, and a decrease in insulin secretion. Recent studies have highlighted the fundamental role of A2a adenosine receptor (A2aR) in potentiation of insulin secretion and proliferation of PBCs. However, possible protective effects of A2aR signaling against ER stress have not been elucidated yet. Thus, in the present study, we aimed to investigate the effects of A2aR activation in MIN6 beta cells undergoing tunicamycin (TM)‐mediated ER stress. A2aR expression and activity were evaluated using real‐time polymerase chain reaction and measurement of the cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), phospho‐protein kinase B or Akt (p‐Akt)/Akt, and phospho‐Cyclic adenosine monophosphate response element‐binding protein/CREB levels in response to a specific agonist (CGS 21680). Survival and proliferation in TM and CGS 21680 cotreated cells were evaluated using 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), annexin V–fluorescein isothiocyanate (FITC)/propidium iodide staining, colony formation, and 5‐bromo‐2′‐deoxyuridine (Brdu) assays. In addition, the effects of A2aR stimulation on insulin secretion were evaluated using the enzyme‐linked immunosorbent assay. B‐cell lymphoma 2 (Bcl‐2), phospho‐eukaryotic Initiation Factor 2α (p‐eIF2α)/eIF2α, growth arrest and DNA‐damage‐inducible 34 (GADD34), X‐box binding protein 1 (XBP‐1), spliced X‐box binding protein 1 (XBP‐1s), immunoglobulin heavy‐chain‐binding protein (BIP), and CCAAT‐enhancer‐binding protein homologous protein (CHOP) levels were evaluated using western blotting. Our results showed a decrease in A2aR expression and p‐Akt/Akt and p‐CREB/CREB levels in TM‐pretreated cells. We also mentioned that CGS 21680 effectively increased cell survival, proliferation, and insulin secretion in TM‐treated cells. The antiapoptotic effects were possibly mediated through Bcl‐2 upregulation. Our western blotting results indicated that A2aR effectively downregulated p‐eIF2α/eIF2α, XBP‐1, XBP‐1s, BIP, and CHOP levels, whereas GADD34 was upregulated. Altogether, the present study revealed that A2aR signaling through PKA/Akt/CREB mediators alleviated TM cytotoxicity effects in MIN6 beta cells. Thus, the stimulation of this receptor was seen as a new approach to control ER stress in the PBC cells.

Background:Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and has antiproliferative effect. However, such effects of DHA have not yet been revealed for bladder cancer cells.Methods:We used as bladder cancer cell lines to examine the effect of DHA on the cell viability, cell apoptosis, and monitoring of mitochondrial membrane potential (ΔΨm) changes. Furthermore, the effect of DHA on the reactive oxygen species (ROS) production and cytochrome c release were also detected. We employed MTT assay to investigate the cell proliferation effect of DHA on the EJ-138 and HTB-9 human bladder cancer cells. Annexin/PI staining, caspase-3 activity assay, Bcl-2/Bax protein expression, mitochondrial membrane potential assay, cytochrome c release, and ROS analysis were used for apoptosis detection.Results:DHA significantly reduced cell viability in a dose-dependent manner. Cytotoxicity of DHA was suppressed by N-acetylcysteine. The growth inhibition effect of DHA was related to the induction of cell apoptosis, which were manifested by annexin V-FITC staining, activation of caspase-3. DHA also increased ROS generation, cytochrome c release, and loss of mitochondrial transmembrane potential (ΔΨm) in cells. In addition, the downregulation of regulatory protein Bcl-2 and upregulation of Bax protein by DHA were also observed.Conclusions:These findings demonstrated that DHA induces apoptosis through mitochondrial signaling pathway. These suggest that DHA may be a potential agent for induction of apoptosis in human bladder cancer cells.

Chronic exposure to high glucose induces endoplasmic reticulum (ER) stress in pancreatic beta cells (PBCs). The previous evidence showed that adenosine modulate PBCs viability and insulin secretion. The aim of this study was to evaluate possible involvement of adenosine in protection of MIN6 β‐cells from Tunicamycin (Tu)‐induced ER stress. MIN6 cells were cotreated with Tu and different concentrations of adenosine. Cell viability, proliferation, and apoptosis were evaluated using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide (MTT), 5‐bromo‐2′‐deoxyuridine (Brdu), and colony formation assays. Caspase‐12 activity was assayed using the fluorometric method. Thioflavin T (ThT) staining was used for the evaluation of protein aggregation. Insulin secretion was evaluated using specific an ELISA kit. Ca2+ mobilization assayed using Fura2/AM probe. BIP, CHOP, XBP‐1, and XBP‐1s expression in both messenger RNA (mRNA) and protein levels were evaluated using the reverse transcription‐polymerase chain reaction (RT‐PCR) and Western blot analysis, respectively. Bcl‐2, p‐eIF2α/eIF2α, and GADD34 levels also determined with Western blot analysis. Adenosine protected MIN6 cells against Tu‐induced ER stress in a dose‐dependent manner and increased their proliferation. Decreased caspase‐12 activity and upregulated Bcl‐2 protein may explain antiapoptotic effects of adenosine. ThT staining indicated an attenuated aggregation of misfolded proteins. Adenosine effectively increased insulin secretion in Tu‐treated cells. BIP, CHOP, XBP1, and sXBP1 expression were decreased significantly in cotreated cells, indicating alleviation of ER stress. However, adenosine potentiated the expression of GADD34 and decreased p‐eIF2α/eIF2α ratio. Adenosine increased cytosolic Ca 2+ levels, which may promote adenosine triphosphate (ATP) synthesis in mitochondria, helping ER to preserve protein hemostasis. Taken together, adenosine upregulated Bcl‐2 and GADD34 to protect PBCs against Tu‐induced apoptosis and increase Insulin secretion.

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Diabetes incidence showed ascending trends in recent years indicating urgent need for new therapeutic agents. Extracellular adenosine signaling showed promising results. However, role of its A3 receptor in pancreatic β-cells proliferation and insulin secretion is not well established. Thus, we aimed to determine its main signaling mediators in MIN6 insulinoma cell line. A3 adenosine receptor (A3AR) expression was confirmed using RT-PCR. Receptor functionality was evaluated by measurements of cAMP, using ELISA kit, and intracellular Ca2+ levels, using Fura 2/AM probe in response to the specific A3AR agonist (Cl- IBMECA). Insulin ELISA kit was used to measure insulin release. Herein, we mentioned that MIN6 cells express active form of A3AR, which decreased cAMP levels with the half maximal effective concentration (EC50) value of 5.61. [Ca2+]i Levels transiently (approximately 120 sec) increased in response to the agonist. Cl-IBMECA increase insulin secretion at 0.01-1 μM, but showed an inhibitory effects at higher concentrations (1-10 μM). Altogether, we found that in MIN6 cells, A3AR, possibly through Ca2+ mediated signaling pathways, potentiated glucose-induced insulin secretion.

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