Fraxetin reduces endometriotic lesions through activation of ER stress, induction of mitochondria-mediated apoptosis, and generation of ROS.

BACKGROUND: Fraxetin, a phytochemical obtained from Fraxinus rhynchophylla, is well known for its anti-inflammatory and anti-fibrotic properties. However, fraxetin regulates the progression of endometriosis, which is a benign reproductive disease that results in low quality of life and infertility. HYPOTHESIS/PURPOSE: We hypothesized that fraxetin may have therapeutic effects on endometriosis and aimed to elucidate the underlying mechanisms of mitochondrial function and tiRNA regulation. STUDY DESIGN: Endometriotic animal models and cells (End1/E6E7 and VK2/E6E7) were used to identify the mode of action of fraxetin. METHODS: An auto-implanted endometriosis animal model was established and the effects of fraxetin on lesion size reduction were analyzed. Cell-based assays including proliferation, cell cycle, migration, apoptosis, mitochondrial function, calcium efflux, and reactive oxygen species (ROS) were performed. Moreover, fraxetin signal transduction was demonstrated by western blotting and qPCR analyses. RESULTS: Fraxetin inhibited proliferation and migration by inactivating the P38/JNK/ERK mitogen-activated protein kinase (MAPK) and AKT/S6 pathways. Fraxetin dissipates mitochondrial membrane potential, downregulates oxidative phosphorylation (OXPHOS), and disrupts redox and calcium homeostasis. Moreover, it triggered endoplasmic reticulum stress and intrinsic apoptosis. Furthermore, we elucidated the functional role of tiRNAHisGTG in endometriosis by transfection with its inhibitor. Finally, we established an endometriosis mouse model and verified endometriotic lesion regression and downregulation of adhesion molecules with inflammation. CONCLUSION: This study suggests that fraxetin is a novel therapeutic agent that targets mitochondria and tiRNAs. This is the first study to demonstrate the mechanisms of tiRNAHisGTG with mitochondrial function and cell fates and can be applied as a non-hormonal method against the progression of endometriosis.

Alpinumisoflavone Activates Disruption of Calcium Homeostasis, Mitochondria and Autophagosome to Suppress Development of Endometriosis.

Alpinumisoflavone is an isoflavonoid extracted from the Cudrania tricuspidate fruit and Genista pichisermolliana. It has various physiological functions, such as anti-inflammation, anti-proliferation, and apoptosis, in malignant tumors. However, the effect of alpinumisoflavone is still not known in chronic diseases and other benign reproductive diseases, such as endometriosis. In this study, we examined the cell death effects of alpinumisoflavone on the endometriosis cell lines, End1/E6E7 and VK2/E6E7. Results indicated that alpinumisoflavone inhibited cell migration and proliferation and led to cell cycle arrest, depolarization of mitochondria membrane potential, apoptosis, and disruption of calcium homeostasis in the endometriosis cell lines. However, the cellular proliferation of normal uterine epithelial cells was not changed by alpinumisoflavone. The alteration in Ca2+ levels was estimated in fluo-4 AM-stained End1/E6E7 and VK2/E6E7 cells after alpinumisoflavone treatment with or without calcium inhibitor, 2-aminoethoxydiphenyl borate (2-APB). The results indicated that a combination of alpinumisoflavone and a calcium inhibitor reduced the calcium accumulation in the cytosol of endometriosis cells. Additionally, alpinumisoflavone decreased oxidative phosphorylation (OXPHOS) in the endometriotic cells. Moreover, protein expression analysis revealed that alpinumisoflavone inactivated AKT signaling pathways, whereas it increased MAPK, ER stress, and autophagy regulatory proteins in End1/E6E7 and VK2/E6E7 cell lines. In summary, our results suggested that alpinumisoflavone could be a promising effective management agent or an adjuvant therapy for benign disease endometriosis.

Inhibitory Effects of 6,8-Diprenylorobol on Endometriosis Progression in Humans by Disrupting Calcium Homeostasis and Mitochondrial Function.

6,8-Diprenylorobol is a flavonoid compound extracted from Cudrania tricuspidata. It has various biological functions, such as inhibiting melanin synthesis and inducting cell death in cancerous cells. In addition, Cudrania tricuspidata is known to be effective in female diseases, and previous studies have shown anticancer effects in cervical cancer, a female reproductive disease. Outside of that, Cudrania tricuspidata has various physiological effects. However, the effect of 6,8-diprenylorobol is not well known in other benign and chronic diseases, even in endometriosis, which commonly arises in the female reproductive tract. In the present study, we determined the inhibitory effects of 6,8-diprenylorobol on the growth of endometriosis VK2/E6E7 and End1/E6E7 cells. Results indicated that 6,8-diprenylorobol suppressed cellular proliferation and increased the disruption of the cell cycle, mitochondrial membrane potential (MMP), generation of reactive oxygen species, and Ca2+ homeostasis in both endometriosis cells. However, the proliferation of normal stromal cells isolated from endometrial tissue was not altered by 6,8-diprenylorobol. The change in Ca2+ levels was estimated in fluo-4- or rhod-2-stained VK2/E6E7 and End1/E6E7 cells after the treatment of the intracellular calcium regulators 2-aminoethoxydiphenyl borate (2-APB) and ruthenium red (RUR) with 6,8-diprenylorobol. A combination of 6,8-diprenylorobol with each regulator decreased the calcium accumulation in endometriosis cells. Furthermore, Western blot analysis indicated that 6,8-diprenylorobol inactivated AKT pathways, whereas it activated P38 MAPK pathways. In addition, 6,8-diprenylorobol decreased mitochondrial respiration, leading to the reduction in ATP production in VK2/E6E7 and End1/E6E7 cells. Collectively, our results suggested that 6,8-diprenylorobol might be a potential therapeutic agent or adjuvant therapy for the management of endometriosis.

Fraxetin induces cell death in colon cancer cells via mitochondria dysfunction and enhances therapeutic effects in 5-fluorouracil resistant cells.

Fraxetin is a natural compound extracted from Fraxinus spp. and has various functions such as antibacterial, antioxidant, neuroprotective, and antifibrotic effects. Although studies have reported its anticancer properties in lung and breast cancer, little is known about colon cancer, the most frequent type of cancer. Thus, we used two colon cancer cell lines, HT29 and HCT116 cells, to investigate whether fraxetin could inhibit the capabilities acquired during tumor development. In this study, fraxetin suppressed cell viability and induced apoptotic cell death in HT29 and HCT116 cells. Furthermore, fraxetin regulated the expression of proteins involved in apoptosis in HT29 and HCT116 cells. Additionally, fraxetin induced reactive oxygen species levels and calcium influx with loss of mitochondrial membrane potential (ΔΨm) and endoplasmic reticulum stress. Moreover, fraxetin induced G2/M arrest and modulated the intracellular signaling pathway, including AKT, ERK1/2, JNK, and P38. Nevertheless, we found no cause-effect correlation between the antiproliferative action of fraxetin and modulation of the phosphorylation state of signaling proteins. Fraxetin-induced inhibitory effect on colon cancer cell viability was synergistic with 5-fluorouracil (5-FU) or irinotecan even in 5-FU resistant-HCT116 cells. Collectively, our results suggest that fraxetin can be effectively used as a therapeutic agent for targeting colon cancer, although it is necessary to further elucidate the relationship between the hallmark capabilities that fraxetin inhibits and the intracellular regulatory mechanism.

Administration of Kyung-Ok-Ko reduces stress-induced depressive behaviors in mice through inhibition of inflammation pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Kyung-Ok-Ko (KOK), a traditional medicinal formula composed of Rehmannia glutinosa (Gaertn.) DC, Poria cocos (Schw.) Wolf, Korean Red Panax ginseng C.A.Mey, and honey, has been used to treat amnesia and dementia. KOK has also been shown to ameliorate transient cerebral global ischemia-induced brain damage, but the antidepressant-like effect of KOK has not been examined. AIM OF THE STUDY: This study examined the antidepressant-like effect of KOK in an immobilization-induced stress mouse and its mechanisms of action. MATERIALS AND METHODS: The animals in the stress group were immobilized for two hours a day for two weeks. KOK at a dose of 1 g/kg/day was administered orally to the stressed mice for two weeks in advance of their immobilization. A forced swimming test was performed to analyze their depressive behaviors. To examine the anti-inflammatory or antioxidative effects of KOK, the murine macrophage cell line, RAW 264.7 cells and human neuroblastoma cell, SH-SY5Y cells, were treated with lipopolysaccharide (LPS) and hydrogen peroxide, respectively. RESULT: The KOK extract showed no significant toxicity when the cells were treated with a KOK extract at 5, 10, 25, 50, and 100 µg/mL. The KOK ethanol extract reduced LPS-induced TNF-α production, inducible nitric oxide (iNOS) mRNA level, and the levels of MAPK and p38 phosphorylation in RAW 264.7 cells. KOK also suppressed H2O2-induced cell death and the production of reactive oxygen species (ROS) in SH-SY5Y cells. In the forced swimming test, KOK induced a decrease in immobility and an increase in climbing activity. Finally, the administration of KOK reversed the up-regulation of IkB-α phosphorylation in the stressed mouse cortex. CONCLUSION: KOK might be useful for the treatment of depression caused by environmental and lifestyle-related stress.

Myricetin inhibits endometriosis growth through cyclin E1 down-regulation in vitro and in vivo.

Endometriosis is a benign gynecological condition prevalent among reproductive-aged women. Although active research and studies have been carried out to discover new drugs, surgery and hormone therapy are still the gold standard for endometriosis treatment. Nowadays, various flavonoids are considered long-term supplements for different diseases. Myricetin, a flavonol, has antiproliferative, anti- or pro-oxidant, and anticancer effects in gynecological diseases. Here, we reveal for the first time, to our knowledge, the antigrowth effects of myricetin in endometriosis. Myricetin inhibited cell proliferation and cell cycle progression of human VK2/E6E7 and End1/E6E7 cells and induced apoptosis, with the loss of mitochondrial membrane potential and accumulation of reactive oxygen species and calcium ions. Additionally, myricetin decreased the activation of AKT and ERK1/2 proteins, whereas it induced p38 activation in both cell lines. Moreover, myricetin decreased lesion size in the endometriosis mouse model via Ccne1 inhibition. Thus, myricetin has antiproliferative effects on endometriosis through cell cycle regulation.

Selection of patients with ovarian cancer who may show survival benefit from hyperthermic intraperitoneal chemotherapy: A systematic review and meta-analysis.

BACKGROUND: The use of hyperthermic intraperitoneal chemotherapy (HIPEC) after cytoreductive surgery has been extensively studied in patients with peritoneal carcinomatosis from various malignancies. However, the effectiveness of HIPEC for ovarian cancer is still controversial. Therefore, we performed this meta-analysis to identify patients with ovarian cancer who can obtain survival benefit from HIPEC. METHODS: Articles regarding HIPEC in the MEDLINE, EMBASE, and Cochrane Library were searched till December 2018. In total, 13 case-control studies and two randomized controlled trials were included in this meta-analysis. We investigated the effect of HIPEC on disease-free survival (DFS) and overall survival (OS), and performed subgroup analyses based on the study design, adjustment of confounding variables, and quality of the study. RESULTS: HIPEC improved both DFS (hazard ratio [HR], 0.603; 95% confidence interval [CI], 0.513-0.709) and OS (HR, 0.640; 95% CI, 0.519-0.789). In cases of primary disease, HIPEC improved DFS (HR, 0.580; 95% CI, 0.476-0.706) and OS (HR, 0.611; 95% CI, 0.376-0.992). Subgroup analyses revealed that HIPEC did not improve OS but improved DFS of patients with residual tumors ≤1 cm or no visible tumors. In cases of recurrent disease, HIPEC was associated with better OS (HR, 0.566; 95% CI, 0.379-0.844) but not with DFS. Subgroup analyses also revealed similar tendencies. However, HIPEC improved DFS of patients with residual tumors ≤1 cm or no visible tumors, while it improved OS of only those with residual tumors ≤1 cm. CONCLUSIONS: HIPEC may improve DFS of patients with ovarian cancer when residual tumors were ≤1 cm or not visible. It may also improve OS of only patients with recurrent disease whose residual tumors were ≤1 cm.

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.

Electromagnetic acupuncture to enhance the effects of manual acupuncture on recovery from muscle fatigue of the quadriceps.

The aim of this study was to investigate a new method of manual acupuncture that used a magnetic field to stimulate only one acupoint vertically. We developed an eight-channel electromagnetic acupuncture (EMA) system that uses a solenoid-type electrode to insert the manual acupuncture needle into a hole in an electrode. We used a manual acupuncture needle for magnetic induction in order to penetrate vertically and deeply into tissues. In order to confirm the usefulness of EMA, we investigated the effects of treatment on muscle fatigue after strenuous knee extension/flexion exercises that had been performed by three groups: the nonstimulation, the manual acupuncture, and the EMA groups. Electromyograms showed that the median frequency (MF) in the EMA group had rapidly recovered after 4 minutes (p = 0.608), but that the peak torque had not recovered to the normal state (p < 0.05). Thus, we confirmed that compared with manual acupuncture, EMA resulted in better recovery from muscle fatigue.

Verification of accuracy and validity of gait phase detection system using motion sensors for applying walking assistive FES.

In this study, we have analysed heel strike (HS) and toe off (TO) of normal individuals and hemiplegic patients, taking advantage of output curves acquired from various sensors, and verified the validity of sensor detection methods and their effectiveness when they were used for hemiplegic gaits. Gait phase detections using three different motion sensors were valid, since they all had reliabilities more than 95%, when compared with foot velocity algorithm. Results showed that the tilt sensor and the gyrosensor could detect gait phase more accurately in normal individuals. Vertical acceleration could detect HS most accurately in hemiplegic patient group A. The gyrosensor could detect HS and TO most accurately in hemiplegic patient groups A and B. The detection of TO from all sensor signals was valid in both the patient groups A and B. However, the vertical acceleration detected HS validly in patient group A and the gyrosensor detected HS validly in patient group B.

Comparison of 2 methods of non-invasive treatment between transcutaneous electrical stimulation and pulsed electromagnetic field stimulation as replacement of invasive manual acupuncture.

The aim of this study was to find the non-invasive optimal alternative method for Manual Acupuncture. Existing researches had reported that Transcutaneous Electrical Acupoint Stimulation (TEAS) was an effective treatment method instead of manual acupuncture. In place of the TEAS, we suggested the Pulsed Electromagnetic Fields (PEMFs). Thus, we designed the PEMFs system which can stimulate only an acupoint. There have been no researches which reported therapeutic effect when stimulating at an identical acupoint by TEAS and PEMFs. Hence, this study investigated the therapeutic effect on the muscle fatigue after the strenuous knee extension/flexion exercise by two stimulations. We selected the stimulation method of both TEAS and PEMFs by using 2Hz biphasic rectangular wave pulse and pulse width 0.2ms. The magnetic flux was the 30.92mT (309.2gauss) at 2 Hz. The electromyogram (EMG) and the maximal voluntary contraction (MVC) at rectus femoris were measured. The Median Frequency (MF) at TEAS group was significantly effective at 6 minutes (p=0.499). The PEMFs group was recovered to the MF rapidly after 4 minutes (p=0.166). The results of the peak torque indicated that both non-stimulation group and TEAS group did not recover to the peak torque at pre-exercise during the recovery period (p<0.05). In contrast, the significant treatment effect of PEMFs group was found after 14 minutes (p=0.135). The results of this study demonstrated that PEMFs were better than TEAS as a non-invasive method to replace the manual acupuncture.

Anti-apoptotic effect of melatonin on preimplantation development of porcine parthenogenetic embryos.

In the present study, we investigated the effect of melatonin on the preimplantation development of porcine parthenogenetic and somatic cell nuclear transfer (SCNT) embryos. Parthenogenetic embryos were cultured in mNCSU-23 supplemented with various concentrations of melatonin for 7 days. The results revealed that 100 pM was the optimal concentration, which resulted in significantly increased cleavage and blastocyst formation rates. Additionally, 100 pM melatonin provided the highest increase in total cell number of blastocysts. Therefore, the subsequent experiments were performed with 100 pM melatonin. ROS level in 2-8 cell stage embryos in the presence or absence of melatonin was evaluated. Embryos cultured with melatonin showed significantly decreased ROS. Blastocysts cultured with melatonin for 7 days were analyzed by the TUNEL assay. It was observed that melatonin not only increased (P < 0.05) the total cell number but also decreased (P < 0.05) the rate of apoptotic nuclei. Blastocysts cultured with melatonin were assessed for the expression of apoptosis-related genes Bcl-xl and Bax, and of pluripotency marker gene Oct-4 by real-time quantitative PCR. Analysis of data showed that the expression of Bcl-xl was higher (1.7-fold) compared to the control while the expression of Bax was significantly decreased relative to the control (0.7-fold) (P < 0.05). Moreover, the expression of Oct-4 was 1.7-fold higher than the control. These results indicated that melatonin had beneficial effects on the development of porcine parthenogenetic embryos. Based on the findings of parthenogenetic embryos, we investigated the effect of melatonin on the development of porcine SCNT embryos. The results also demonstrated increased cleavage and blastocyst formation rates, and the total cell numbers in blastocysts were significantly higher when the embryos were cultured with melatonin. Therefore, these data suggested that melatonin may have important implications for improving porcine preimplantation SCNT embryo development.