Whasun Lim

Apigenin induces ROS-dependent apoptosis and ER stress in human endometriosis cells

Apigenin is a plant‐derived flavonoid having antiproliferative, anti‐inflammatory, and anti‐angiogenic properties in chronic and metabolic diseases, and cancers. However, the functional role of apigenin remains to be identified in human endometriosis that is a benign inflammatory disease causing infertility, dysmenorrhea, dyspareunia, and chronic abdominal or pelvic pain. In the present study, we determined the effects of apigenin on two well‐established human endometriosis cell lines (VK2/E6E7 and End1/E6E7). Apigenin reduced proliferation and induced cell cycle arrest and apoptosis in the both endometriosis cell lines. In addition, it disrupted mitochondrial membrane potential (MMP) which was accompanied by an increase in concentration of calcium ions in the cytosol and in pro‐apoptotic proteins including Bax and cytochrome c in the VK2/E6E7 and End1/E6E7 cells. Moreover, apigenin treated cells accumulated excessive reactive oxygen species (ROS), and experienced lipid peroxidation and endoplasmic reticulum (ER) stress with activation of the unfolded protein response (UPR) regulatory proteins. Furthermore, the apigenin‐induced apoptosis in endometriosis cells was regulated via the ERK1/2, JNK, and AKT cell signaling pathways. Taken together, apigenin is a potential novel therapeutic agent to overcome current limitations in the treatment to endometriosis.

Myricetin suppresses invasion and promotes cell death in human placental choriocarcinoma cells through induction of oxidative stress

Myricetin is a bioactive compound found in a variety of vegetables and fruits, and its anti-cancer effects are well known. In this study, we confirmed that myricetin reduced proliferation of two choriocarcinoma cell lines (JAR and JEG-3) and also promoted apoptosis and regulated cell cycle progression in a dose-dependent manner in JAR and JEG-3 cells. In addition, we found that invasive and pro-angiogenic properties of malignant JAR and JEG-3 trophoblast cells were attenuated by myricetin treatment via MAPK and PI3K/AKT signaling pathways. In addition, we found that ROS production, lipid peroxidation, glutathione depletion, and loss of mitochondrial membrane potentials were enhanced in JAR and JEG-3 cells treated with myricetin. Moreover, myricetin augmented cytosolic Ca2+ release from the endoplasmic reticulum associated with modulation of ER stress in JAR and JEG-3 cells. Our results also revealed that myricetin had synergistic antiproliferative effects with current chemotherapeutics, etoposide and cisplatin, on choriocarcinoma cells. Collectively, results of the present study provide strong evidence for the potential of myricetin to be an effective therapeutic for the prevention of human placental choriocarcinomas.

Silibinin stimluates apoptosis by inducing generation of ROS and ER stress in human choriocarcinoma cells

Silibinin is a flavonolignan extracted from seeds of milk thistles. Traditionally, it has been used as a therapeutic agent for liver disorders, and now it is well‐known for its anti‐cancer effects. However, studies on anti‐cancer effects of silibinin on choriocarcinoma are very limited. Therefore, we performed proliferation and apoptosis assays to determine effects of silibinin on the viability of human choriocarcinoma (JAR and JEG3) cells. Our results showed that silibinin significantly inhibited proliferation and induced apoptosis in both JAR and JEG3 cells, and significantly increased reactive oxygen species (ROS) and lipid peroxidation. Moreover, silibinin disrupted mitochondrial function by inducing permeabilization of mitochondrial membrane potential and calcium ion efflux in JAR and JEG3 cells. Furthermore, silibinin‐induced apoptosis in choriocarcinoma cells via AKT, mitogen‐activated protein kinases (MAPK) and unfolded protein response (UPR) signal transduction. Collectively, our results suggest that silibinin is a novel therapeutic agent or dietary supplement for management of human placental choriocarcinomas.

Cell-specific expression and signal transduction of C-C motif chemokine ligand 2 and atypical chemokine receptors in the porcine endometrium during early pregnancy

&NA; Chemokines and atypical chemokine receptors (ACKRs; also known as chemokine decoy receptors) play an important role in reproductive immunology by recruiting leukocytes during early pregnancy. The aim of this study was to determine the expression of C‐C motif chemokine ligand 2 (CCL2) and ACKRs in the endometrium during estrous cycle and early pregnancy, and to investigate the functional effects of CCL2 on porcine uterine luminal epithelial (pLE) cells. Our results indicated that CCL2, ACKR1, ACKR3, and ACKR4 were strongly detected in the glandular and luminal epithelium of the endometrium during early pregnancy compared to that in non‐pregnant pigs. Recombinant CCL2 improved pLE cell proliferation via activation of the PI3K and MAPK pathways and suppression of endoplasmic reticulum (ER) stress by reducing the expression of ER stress regulatory genes. Collectively, these results provide novel insights into CCL2‐mediated signaling mechanisms in the porcine endometrium at the maternal‐fetal interface during early pregnancy. HighlightsCCL2‐ACKR system is regulated in the porcine uterine endometrium.CCL2 induces the proliferation and cell cycle progression of porcine uterine cells.CCL2 activates PI3K/AKT and MAPK pathways for cellular proliferation in the uterus.CCL2 reduces ER stress regulatory gene expression in porcine luminal epithelia cells.CCL2 may play an important role in maternal‐fetal interaction during early pregnancy.

Decanoic acid suppresses proliferation and invasiveness of human trophoblast cells by disrupting mitochondrial function

&NA; Decanoic acid (DA) is a medium‐chain fatty acid used in the manufacture of various products including plastics, cosmetics, and lubricants. In addition to antiviral and antibacterial effects, DAs, reported biological activities include regulation of signaling pathways and redox homeostasis in various human cell types. The influence of DA on functional properties of human trophoblasts, including proliferation, invasion and apoptosis is currently unknown. In the present study, we evaluated the anti‐proliferative and anti‐invasive effects of DA on the human trophoblast cell line HTR8/SVneo. In addition, DA induced oxidative stress, as evidenced by generation of reactive oxygen species (ROS) and induction of lipid peroxidation (LPO). This oxidative stress was accompanied by activation of the mitochondria‐dependent apoptotic pathway in HTR8/SVneo cells. We also observed elevated mitochondrial Ca2 +, and loss of mitochondrial membrane potential in response to DA treatment. Chelation of mitochondrial Ca2 + using BAPTA‐AM rescued cellular proliferation suppressed by DA. We also verified that signaling proteins including AKT, P70S6K, S6, and ERK1/2 and their targets were significantly reduced in HTR8/SVneo cells by DA treatment. Pre‐treatment of cells with selective inhibitors of AKT (LY294002) and ERK1/2 (U0126) revealed that the AKT and ERK1/2 signaling pathways regulated by DA displayed cross‐talk in HTR8/SVneo cells. Collectively, these results suggest that personal products containing DA will have harmful effects on human trophoblasts, and could cause implantation and placentation failure during early pregnancy. HighlightsDecanoic acid inhibits proliferation and invasion of human trophoblasts.Decanoic acid blocks AKT and ERK1/2 signaling pathways in human trophoblasts.Decanoic acid‐induced oxidative stress results in mitochondrial membrane disruption.

Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells

Structural and functional development of the mammary gland is constant in the mammary gland life cycle. Eph receptors and their ligands, ephrins, control events through cell‐to‐cell interactions during embryonic development, and adult tissue homeostasis; however, little information on participation of ephrin A1, a representative ligand of the Eph receptor, in the development and function of normal mammary glands is known. In this study, we demonstrated functional effects of the ephrin A1‐Eph system and mechanisms of its action on bovine mammary epithelial (MAC‐T) cells. The in vitro cultured MAC‐T cells expressed the ephrin A1 ligand and EphA1, A2, A4, A7, and A8 among the eight members of the Eph A family. Our results revealed that ephrin A1 induced MAC‐T cell cycle progression and stimulated cell proliferation with abundant expression of nucleic PCNA and cyclin D1 proteins. Additionally, ephrin A1 induced activation of intracellular signaling molecules involved in PI3u2009K/AKT and MAPK signaling, and the proliferation‐stimulating effect of ephrin A1 was mediated by activation of these pathways. Furthermore, ephrin A1 influenced expression and activation of various ER stress‐related proteins and protected MAC‐T cells from stress‐induced cell death. Finally, ephrin A1 alleviated LPS‐induced cell death through down‐regulation of inflammatory cytokines. In conclusion, the results of this study suggest that the Eph A‐ephrin A1 system is a positive factor in the increase and maintenance of epithelial cells in mammary glands of cows; the signaling system contributes to development, remodeling, and functionality of normal mammary glands and could overcome mastitis in cows and other mammals.

Chrysin attenuates progression of ovarian cancer cells by regulating signaling cascades and mitochondrial dysfunction

Chrysin is mainly found in passion flowers, honey, and propolis acts as a potential therapeutic and preventive agent to inhibit proliferation and invasion of various human cancer cells. Although chrysin has anti‐carcinogenic effects in several cancers, little is known about its functional roles in ovarian cancer which shows poor prognosis and chemoresistance to traditional therapeutic agents. In the present study, we investigated functional roles of chrysin in progression of ovarian cancer cells using ES2 and OV90 (clear cell and serous carcinoma, respectively) cell lines. Results of the current study demonstrated that chrysin inhibited ovarian cancer cell proliferation and induced cell death by increasing reactive oxygen species (ROS) production and cytoplasmic Ca2+ levels as well as inducing loss of mitochondrial membrane potential (MMP). Moreover, chrysin activated mitogen‐activated protein kinase (MAPK) and phosphoinositide 3‐kinase (PI3K)/AKT pathways in ES2 and OV90 cells in concentration‐response experiments. Collectively, our results led us to propose that chrysin‐induced apoptotic events are mediated by the activation of PI3K and MAPK pathways in human ovarian cancer cells.

Naringenin induces mitochondria-mediated apoptosis and endoplasmic reticulum stress by regulating MAPK and AKT signal transduction pathways in endometriosis cells

STUDY QUESTIONnDoes the flavonoid naringenin inhibit proliferation of human endometriosis cells?nnnSUMMARY ANSWERnNaringenin suppresses proliferation and increases apoptosis via depolarization of mitochondrial membrane potential and generation of reactive oxygen species (ROS) in human endometriosis cells.nnnWHAT IS KNOWN ALREADYnFor management of endometriosis, hormonal therapy is commonly used to decrease production of estrogens by the ovaries, but that has limitations including undesirable side effects with long-term therapies. To overcome these limitations, it is important to discover novel compounds which have no adverse effects, but inhibit expression of target molecules involved in the pathogenesis of endometriosis.nnnSTUDY DESIGN SIZE, DURATIONnWell-established endometriosis cell lines (VK2/E6E7 and End1/E6E7) were purchased from the American Type Culture Collection. Effects of naringenin on VK2/E6E7 and End1/E6E7 cells were assessed in diverse assays in a dose- and time-dependent manner.nnnPARTICIPANTS/MATERIALS, SETTING, METHODSnEffects of naringenin on viability, apoptosis (Annexin V expression, propidium iodide staining, TUNEL and invasion assays), mitochondria-mediated apoptosis, production of ROS and endoplasmic reticulum (ER) stress proteins of VK2/E6E7 and End1/E6E7 cells were determined. Signal transduction pathways in VK2/E6E7 and End1/E6E7 cells in response to naringenin were determined by western blot analyses.nnnMAIN RESULTS AND THE ROLE OF CHANCEnIn the present study, we demonstrated that naringenin suppressed proliferation and increased apoptosis through depolarization of mitochondrial membrane potential and inducing pro-apoptotic proteins, Bax and Bak, in both endometriosis cell lines. In addition, naringenin increased ROS, ER stress, through activation of eIF2α and IRE1α, GADD153 and GRP78 proteins in a dose-dependent manner. Furthermore, the induction of apoptosis by naringenin involved activation of MAPK and inactivation of PI3K pathways in VK2/E6E7 and End1/E6E7 cells.nnnLIMITATIONS REASONS FOR CAUTIONnLack of in vivo animal studies is a major limitation of this research. Effectiveness of naringenin to induce apoptosis of human endometriosis cells requires further investigation.nnnWIDER IMPLICATIONS OF THE FINDINGSnOur results suggest that naringenin is a promising therapeutic compound for treatment of endometriosis in women.nnnSTUDY FUNDING/COMPETING INTEREST(S)nThis work was supported by grants from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (No. HI15C0810 awarded to G.S. and HI17C0929 awarded to W.L.). The authors declare that there are no conflicts of interest.

Propyl gallate induces cell death and inhibits invasion of human trophoblasts by blocking the AKT and mitogen-activated protein kinase pathways

Propyl gallate (PG) is an antioxidant widely used in food additives, cosmetics, adhesives, and lubricants. PG protects the oils in food products from reacting with hydrogen peroxide and oxygen free radicals, thus preventing spoilage. It is known to have both protective and cytotoxic effects on various cells, but its effects on human trophoblasts remain unclear. Therefore, we investigated the effects of PG on proliferation, apoptosis, and invasiveness of human trophoblasts using an immortalized HTR8/SVneo cell line. We found that PG significantly reduced proliferation of and induced apoptosis in HTR8/SVneo cells. In addition, the invasiveness of HTR8/SVneo cells was attenuated in response to PG. Signaling pathways including the PI3K/AKT and MAPK pathways involved in the proliferation and invasiveness of human trophoblasts were regulated by PG treatment in HTR8/SVneo cells. We confirmed that mitochondrial membrane disruption and Ca2+ overload were markedly elevated in response to PG treatment, suggesting that PG-induced apoptosis is closely related to mitochondrial dysfunction and further PG-induced apoptosis in HTR8/SVneo cells is related to endoplasmic reticulum (ER) stress. Collectively, these results indicate that PG exerts harmful effects on human trophoblasts; therefore, exposure to PG in early pregnancy is predicted to cause abnormal implantation and placental development.

Differential expression and functional roles of chemokine (C-C motif) ligand 23 and its receptor chemokine (C-C motif) receptor type 1 in the uterine endometrium during early pregnancy in pigs

Abstract Many chemokines expressed by cells of the uterine endometrium of mammals are involved in cell‐cell interactions. However, little is known about expression and functional roles of chemokine (C‐C motif) ligand 23 (CCL23) in the uterine endometrium. Results of this study demonstrated that CCL23 and its receptor, chemokine (C‐C motif) receptor type 1 (CCR1), are up‐regulated in porcine endometria during pregnancy. CCL23 and CCR1 mRNAs were strongly expressed in endometrial glandular (GE) and luminal (LE) epithelial cells. Treatment of porcine uterine luminal epithelial (pLE) cells with recombinant CCL23 increased the abundances of PCNA and cyclin D1, and enhanced proliferation and cell cycle progression in pLE cells. CCL23 also stimulated phosphorylation of cell signaling molecules including AKT and MAPKs in pLE cells. Furthermore, ER stress‐related molecules were reduced by CCL23. These results suggest that CCL23‐CCR1 signaling is important for endometrial development and establishment of pregnancy in pigs. HighlightsCCL23‐CCR1 system is up‐regulated in the uterine endometrium during early pregnancy.CCL23 induces the proliferation and cell cycle progression of pLE cells.CCL23 activates intracellular signaling pathways including PI3K/AKT and MAPK.CCL23 reduces tunicamycin‐induced ER stress in pLE cells.CCL23‐CCR1 is important for development of the endometrium during early pregnancy.