Metformin increased cellular ROS levels in AsPC-1 pancreatic cancer cells, with minimal effect in HDF, human primary dermal fibroblasts. Metformin reduced cellular ATP levels in HDF, but not in AsPC-1 cells. Metformin increased AMPK, p-AMPK (Thr172), FOXO3a, p-FOXO3a (Ser413), and MnSOD levels in HDF, but not in AsPC-1 cells. p-AMPK and p-FOXO3a also translocated from the cytosol to the nucleus by metformin in HDF, but not in AsPC-1 cells. Transfection of si-FOXO3a in HDF increased ROS levels, while wt-FOXO3a-transfected AsPC-1 cells decreased ROS levels. Metformin combined with apigenin increased ROS levels dramatically and decreased cell viability in various cancer cells including AsPC-1 cells, with each drug used singly having a minimal effect. Metformin/apigenin combination synergistically decreased mitochondrial membrane potential in AsPC-1 cells but to a lesser extent in HDF cells. Metformin/apigenin combination in AsPC-1 cells increased DNA damage-, apoptosis-, autophagy- and necroptosis-related factors, but not in HDF cells. Oral administration with metformin/apigenin caused dramatic blocks tumor size in AsPC-1-xenografted nude mice. Our results suggest that metformin in cancer cells differentially regulates cellular ROS levels via AMPK-FOXO3a-MnSOD pathway and combination of metformin/apigenin exerts anticancer activity through DNA damage-induced apoptosis, autophagy and necroptosis by cancer cell-specific ROS amplification.

In the present study, the antinociceptive profiles of coumarin were examined in ICR mice. Coumarin administered orally (from 1 to 10 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. Duration of antinociceptive action of coumarin maintained at least for 60 min. But, the cumulative response time of nociceptive behaviors induced by a subcutaneous (s.c.) formalin injection, intrathecal (i.t.) substance P (0.7 µg) or glutamate (20 µg) injection was not affected by coumarin. 1) Coumarins form a large group of plant polyphenols. Thus far about 1500 coumarin derivatives have been isolated from plants.2) They occur ubiquitously in the plant kingdom, and coumarin derivatives show a chemotaxonomical tendency to accumulate in large amounts in Rutaceae and Apiaceae. 3) Coumarin has been found to exhibit a wide range of bioactivities, such as hemoprevention against pathogens, 4) herbivores, 5) and abiotic stresses, 6) suggesting that physiological roles of coumarins for plants for the adaption to environmental stresses. Some coumarin derivatives are also known to act beneficially on human health due to their therapeutic effects such as inhibitory activities against various tumor cells, 7,8) mycobacteria, 9) antioxidant, 10,11) antihyperglycemic, 12) antifungal, 13) and antiasthmatic 14) which have been extensively studied in the medical and pharmaceutical fields for the treatment of diseases of human.Previous studies have demonstrated that some coumarins exert an antinociceptive action.15,16) However, exact antinociceptive profiles and mechanism of coumarin have not been well characterized. Thus, we, in the current study, tried to characterize antinociceptive profiles and mechanisms of coumarin in several pain models. Experimental Animals Male iCR mice (MJ Co., Seoul, Korea) weighing 20-25 g were used for all the experiments. Animals were housed 5 per cage in a room maintained at 22± 0.5°C with an alternating 12 h light-dark cycle. Food and water were available ad libitum. The animals were allowed to adapt to the laboratory for at least 2 h before testing and were only used once. experiments were performed during the light phase of the cycle (10:00-17:00). MATeRiALS And MeTHOdSDrug Administration Oral administration was performed with gage in a volume of 500 µL/25 g body weight. intraperitoneal (i.p.) injection was conducted to unanesthesized mice with volume of 250 µL. The intracerebroventricular (i.c.v.) administration followed the method described by Haley.17) The intrathecal (i.t.) administration was performed following the method of Hylden and Wilcox 18,19) using a 30-gauge needle connected to a 25 µL Hamilton syringe with polyethylene tubing. The i.c.v. and i.t. injection volumes were 5 µL and the injection sites were verified by injecting a similar volume of 1% methylene blue solution and determining the distribution of the injected dye in the ventricular space or in the spinal cord. The dye injected i.c.v. was found to be distributed through the v...

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The antinociceptive effects of oleanolic acid were examined in ICR mice. Oleanolic acid administered orally (1, 5 and 10 mg/kg) showed an antinociceptive effect in a dose-dependent manner as measured in the acetic acid-induced writhing test. In the time- course study, duration of antinociceptive action of oleanolic acid maintained at least for 60 min. In addition, the cumulative nociceptive response time for intraplantar formalin injection (2nd phase), intrathecal injection of substance P (0.7 μg) or glutamate (20 μg) was diminished by oleanolic acid. Intraperitoneal (i.p.) pretreatment with naloxone (opioid receptor antagonist) or methysergide (5-HT serotonergic receptor antagonist) attenuated antinociceptive effect induced by oleanolic acid in the writhing test. However, yohimbine (adrenergic receptor antagonist) did not affect antinociception induced by oleanolic acid. The results indicate that oleanolic acid shows an antinociceptive property in various pain models such as writhing, formalin, substance P and glutamate pain tests. Furthermore, this antinociceptive effect of oleanolic acid may be mediated by opioidergic and serotonergic receptors, but not adrenergic receptors.