Sunwoo Park

Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis via the PI3K/AKT and ERK1/2 MAPK Pathways

Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat, and sprouts. It has a variety of biological properties including anti‐oxidant, anti‐mutagenic, anti‐carcinogenic, anti‐inflammatory, anti‐proliferative, and anti‐spasmodic effects. Based on epidemiological and case‐control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti‐cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK, and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose‐dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti‐proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. J. Cell. Physiol. 231: 2690–2699, 2016.

Naringenin-Induced Apoptotic Cell Death in Prostate Cancer Cells Is Mediated via the PI3K/AKT and MAPK Signaling Pathways

Prostate cancer is the most common cancer in men and the second most common cause of cancer‐related deaths in men. Although, various drugs targeting the androgen receptor are normally used, the patients frequently undergo recurrence of the disease. To overcome these limitations, natural compounds have been researched for evidence that they suppress progression and metastasis of various cancer cells. In the present study, we investigated effects of naringenin, a natural anti‐oxidant flavonoid derived from citrus, on prostate cancer cells (PC3 and LNCaP). Results of present study with PC3 and LNCaP cells revealed that naringenin inhibited proliferation and migration, while inducing apoptosis and ROS production by those cells. In addition, naringenin‐induced loss of mitochondrial membrane potential and increased Bax and decreased Bcl‐2 proteins in PC3 cells, but not LNCaP cells. In a dose‐dependent manner, naringenin decreased phosphorylation of ERK1/2, P70S6K, S6, and P38 in PC3 cells, and reduced phosphorylation of ERK1/2, P53, P38, and JNK proteins in LNCaP cells. However, naringenin activated phosphorylation of AKT in both PC3 and LNCaP cells. Then, targeted signaling proteins associated with viability of PC3 and LNCaP cells were analyzed using pharmacological inhibitors of AKT and ERK1/2 cell signaling pathways. Moreover, we compared the apoptotic effects of naringenin and paclitaxel alone and in combination to find that naringenin enhanced the efficiency of paclitaxel to suppress progression of prostate cancer cell lines. Collectively, these results indicate that naringenin is a potential chemotherapeutic agent for treatment of prostate cancer. J. Cell. Biochem. 118: 1118–1131, 2017.

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.

Inhibitory Effects of Quercetin on Progression of Human Choriocarcinoma Cells Are Mediated Through PI3K/AKT and MAPK Signal Transduction Cascades

As a major dietary flavonol, quercetin mitigates proliferation and progression of cancer due to its anti‐angiogenic, anti‐inflammatory, anti‐oxidant, and apoptotic biological effects on cells. Although its apoptotic effects have been reported for various cancers, little is known of the functional role of quercetin in gestational choriocarcinoma. Results of the present study indicated that quercetin reduced proliferation and induced cell death in two choriocarcinoma cell lines, JAR and JEG3 cells, with an increase in the sub‐G1 phase of the cell cycle. In addition, quercetin induced mitochondrial dysfunction significantly reduced mitochondrial membrane potential (MMP) and increased production of reactive oxygen species (ROS) in both JAR and JEG3 cells. Further, quercetin inhibited phosphorylation of AKT, P70S6K and S6 proteins whereas, it increased phosphorylation of ERK1/2, P38, JNK and P90RSK proteins in JAR and JEG3 cells. The decrease in viability of choriocarcinoma cells treated with quercetin was confirmed by using combinations of quercetin and pharmacological inhibitors of the PI3K and MAPK signaling pathways. Classical chemotherapeutic agents, cisplatin (a platinum‐based drug) and paclitaxel (a taxene‐based drug), inhibited proliferation of JAR and JEG3 cells, and when combined with quercetin, the antiproliferative effects of cisplatin and paclitaxel were enhanced for both choriocarcinoma cell lines. Collectively, these results suggest that quercetin prevents development of choriocarcinoma and may be a valuable therapeutic agent for treatment of choriocarcinoma through its regulation of PI3K and MAPK signal transduction pathways. J. Cell. Physiol. 232: 1428–1440, 2017.

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

STUDY QUESTION Does the flavonoid naringenin inhibit proliferation of human endometriosis cells? SUMMARY ANSWER Naringenin suppresses proliferation and increases apoptosis via depolarization of mitochondrial membrane potential and generation of reactive oxygen species (ROS) in human endometriosis cells. WHAT IS KNOWN ALREADY For 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. STUDY DESIGN SIZE, DURATION Well-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. PARTICIPANTS/MATERIALS, SETTING, METHODS Effects 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. MAIN RESULTS AND THE ROLE OF CHANCE In 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. LIMITATIONS REASONS FOR CAUTION Lack 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. WIDER IMPLICATIONS OF THE FINDINGS Our results suggest that naringenin is a promising therapeutic compound for treatment of endometriosis in women. STUDY FUNDING/COMPETING INTEREST(S) This 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.

Myricetin treatment induces apoptosis in canine osteosarcoma cells by inducing DNA fragmentation, disrupting redox homeostasis, and mediating loss of mitochondrial membrane potential

Canine osteosarcoma is an aggressive primary bone tumor that shows metastasis to distal regions and is associated with a high mortality rate. However, the pathophysiological mechanisms of canine osteosarcoma are not well characterized. In addition, development of prognostic factors and novel therapeutic agents is necessary to efficiently treat osteosarcoma. Therefore, we studied the effects of myricetin, an antioxidant found in berries, nuts, teas, wine, and vegetables, on apoptosis and signal transduction in the canine osteosarcoma cell lines, D‐17 and DSN. Results of the present study demonstrated that treatment with myricetin decreased cell proliferation and DNA replication, while it increased apoptotic DNA fragmentation in D‐17 and DSN cells. In addition, it increased generation of ROS, lipid peroxidation, and depolarization of MMP in both D‐17 and DSN cells. Myricetin treatment activated phosphorylation of AKT, p70S6K, ERK1/2, JNK, and p90RSK in canine osteosarcoma cells. Moreover, inhibition of PI3K and MAPK using LY294002, U0126, or SP600125, in addition to myricetin treatment, effectively suppressed cell proliferation compared to treatment with myricetin or each inhibitor alone. Therefore, we concluded that myricetin may be a potentially effective and less toxic therapeutic agent to prevent and control progression of canine osteosarcoma.

The O-methylated isoflavone, formononetin, inhibits human ovarian cancer cell proliferation by sub G0/G1 cell phase arrest through PI3K/AKT and ERK1/2 inactivation

Formononetin is an isoflavone that is extracted from red clovers or soy. It has anti‐oxidant, anti‐proliferative, and anti‐tumor effects against cells in various diseases. Several cohort studies have indicated that phytoestrogen intake, including formononetin, could reduce the risk of various carcinogenesis. In fact, many case‐control studies have indicated the potential value of flavonoids as drug supplements in the treatment of many cancer patients. However, the toxic effects and the anti‐cancer mechanism of formononetin in ovarian cancer are unknown. We investigated the toxicological mechanism of formononetin in ES2 and OV90 ovarian cancer cells. Formononetin suppressed cell proliferation through sub G0/G1 phase arrest and increased apoptosis in both cell lines. Furthermore, it induced loss of mitochondrial membrane potential and generation of reactive oxygen species in ES2 and OV90 cells. The formononetin‐mediated regulation of cell proliferation and apoptosis involved decreased phosphorylation of ERK1/2, P90RSK, AKT, P70S6K, and S6 proteins, and increased phosphorylation of P38 protein in ES2 and OV90 cells. Co‐treatment of formononetin with pharmacological inhibitors (LY294002 or U0126) revealed additional anti‐proliferative effects on the two human ovarian cancer cell types. Conclusively, the results indicate the potential value of formononetin as an anti‐cancer agent in human ovarian cancer.

Naringenin suppresses growth of human placental choriocarcinoma via reactive oxygen species-mediated P38 and JNK MAPK pathways

BACKGROUND Human placental choriocarcinoma is a gestational trophoblastic tumor with high rates of metastasis and reoccurrence. However, some patients with choriocarcinoma are chemoresistance to conventional chemotherapeutic agents. HYPOTHESIS Naringenin increases apoptosis in human placental choriocarcinoma cells. METHODS We investigated the effects of naringenin on proliferation and migration of JAR and JEG3 cells, and performed TUNEL and Annexin V/PI staining assays to examine apoptotic effects of naringenin on both cells. In addition, we studied the loss of mitochondrial membrane potential (MMP) and the production of mitochondrial reactive oxygen species (ROS) to determine the specific reason for apoptosis of choriocarcinoma cells being mediated via mitochondria. Consistent with the induction of production of ROS by naringenin in both choriocarcinoma cell lines, we investigated lipid peroxidation and glutathione levels in both JAR and JEG3 cells since both are affected by ROS. We next determined dose-dependent effects of naringenin and its pharmacological inhibitors on signal transduction pathways in JAR and JEG3 cells by western blot analyses. RESULTS Naringenin reduced viability and migratory functions of both cell lines, and increased mitochondria related apoptosis induced by ROS and lipid peroxidation, decreased glutathione and decreased mitochondrial membrane potential MMP in a dose-dependent manner. We also determined naringenin activated phosphorylation of ERK1/2, P38, JNK and P70S6K in JAR and JEG3 cells in a dose-response manner. Although naringenin induced phosphorylation of AKT proteins in JAR cells, it suppressed phosphorylation of the protein in JEG3 cells. In addition, we confirmed the mechanism of naringenin-induced cell signaling by using a combination of naringenin and pharmacological inhibitors of the PI3K and MAPK pathways, as well as a ROS inhibitor in JAR and JEG3 cell lines. CONCLUSIONS Collectively, results of this study indicate that naringenin is a potential therapeutic molecule with anti-cancer effects on choriocarcinoma cells by inducing generation of ROS and activation of the MAPK pathways.

Chrysophanol selectively represses breast cancer cell growth by inducing reactive oxygen species production and endoplasmic reticulum stress via AKT and mitogen-activated protein kinase signal pathways

&NA; Breast cancer is a leading lethal gynecological cancer. Although many tumor markers and target genes have been studied in breast cancer, its incidence is increasing. Recently, the therapeutic effects of natural phytochemicals have been studied in various cancers as adjuvants. Chrysophanol is an anti‐inflammatory, anti‐angiogenetic, and anti‐tumor anthraquinone but has not been widely studied in cancers. Here, we verified the anti‐cancer effects and cellular mechanism of chrysophanol in human breast cancer cells (BT‐474 and MCF‐7). Chrysophanol selectively inhibited cell proliferation and induced apoptosis of breast cancer cells but not of normal mammary ductal epithelial cells, MCF‐12A. Additionally, chrysophanol increased loss of mitochondrial membrane potential and cytosolic calcium levels to activate pro‐apoptotic proteins, Bax, Bak, and cytochrome c, in both cell lines. Reactive oxygen species (ROS) overproduction by chrysophanol resulted in endoplasmic reticulum (ER) stress, leading to an increase in PERK, eIF2&agr;, GADD153, and IRE1&agr; levels in BT‐474 and MCF‐7 cells. These ER stress proteins increased by chrysophanol were repressed by co‐treatment with N‐acetyl‐L‐cysteine, an ROS inhibitor. Western blotting showed that chrysophanol down‐regulated ERK1/2, AKT, P70S6K, and S6 in both cell lines. However, P38 and JNK activities decreased in BT‐474 cells and increased in MCF‐7 cells. Additionally, co‐treatment with ERK1/2 (U0126) or an AKT inhibitor (LY294002) plus chrysophanol reduced cell proliferation, whereas P38 (SB203580) and a JNK inhibitor (SP600125) showed synergic effects only in BT‐474 cell lines. These results show that chrysophanol has anti‐cancer effects on human breast cancer cells, specifically through mitochondrial apoptosis and ER stress induction. HighlightsChrysophanol inhibits proliferation and invasiveness of human breast cancer cells.Anti‐cancer effects of chrysophanol were similar to commercial anticancer drugs.Chrysophanol blocks PI3K/AKT and MAPK pathways in human breast cancer cells.Toxicity of chrysophanol is accompanied with mitochondria‐dependent apoptosis.

Sideroxylin (Callistemon lanceolatus) suppressed cell proliferation and increased apoptosis in ovarian cancer cells accompanied by mitochondrial dysfunction, the generation of reactive oxygen species, and an increase of lipid peroxidation

Sideroxylin is a C‐methylated flavone isolated from Callistemon lanceolatus and exerts antimicrobial activity against Staphylococcus aureus. However, the anticancer effects of sideroxylin and its intracellular signaling mechanisms have not yet been identified. Results of our study showed that sideroxylin decreased cell proliferation and increased apoptosis, causing DNA fragmentation, depolarization of the mitochondrial membrane, the generation of reactive oxygen species, and an increase of lipid peroxidation in ovarian cancer cells (ES2 and OV90 cells). Additionally, sideroxylin activated the phosphorylation of ERK1/2, JNK, P38, and MAPK proteins and the use of LY294002, U0126, SB203580, and SP600125 to block their phosphorylation, respectively, in ES2 and OV90 cells. Collectively, the results of present study indicated that sideroxylin was a novel therapeutic agent to combat the proliferation of ovarian cancer cells through the induction of mitochondrial dysfunction and the activation of PI3 K and MAPK signal transduction.