• Molecules and Cells
• /
• v.38 no.2
• /
• pp.138-144
• /
• 2015

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells. Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death. Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.

• Clinical and Experimental Reproductive Medicine
• /
• v.49 no.4
• /
• pp.239-247
• /
• 2022

Objective: Asafoetida is a gum derived from Ferula assa-foetida, which is used in traditional Iranian medicine to treat some reproductive system disorders. The effects of asafoetida on ovarian tissue, expression of certain genes associated with polycystic ovary syndrome (PCOS), and levels of liver, kidney, and blood cell factors after treatment in a rat model were investigated. Methods: Thirty rats were divided into five groups: normal, polycystic, and treatment with three doses of asafoetida (12.5, 25, and 50 mg/kg for 3 weeks after PCOS induction). PCOS was induced by letrozole at a dose of 1 mg/kg administered orally for 3 weeks. Blood samples were taken, and the ovaries were removed and prepared for histomorphometric examination. Liver and kidney parameters were measured. The mRNA expression levels of luteinizing hormone receptor, CYP11A1, adenosine monophosphate-activated protein kinase, adiponectin, and adiponectin receptors 1 and 2 were also measured by real-time polymerase chain reaction. Results: The levels of liver, kidney, and blood parameters did not significantly differ between the treatment groups and the control group. At doses of 25 and 50 mg/kg, ovarian histopathology, especially the thicknesses of the theca and granulosa layers, was significantly improved relative to the PCOS group. The expression of target genes also improved in the 25 and 50 mg/kg treatment groups. Conclusion: Asafoetida can be used to treat PCOS as a complementary approach to conventional therapies. Asafoetida appears to act by regulating and activating metabolic and ovarian cycle enzymes.

• The Korean Journal of Pharmacology
• /
• v.30 no.1
• /
• pp.11-18
• /
• 1994

Cyclic adenosine 3'5'-monophosphate (cyclic AMP) has been frequently accepted as an intracellular messenger for receptor-mediated action of opioids. In this experiment, it was designed to determine the interaction of dopaminergic and opioidergic system in the mouse striatum in normal and chronic haloperidol treated groups. Haloperidol 750ug/kg I.P. for 10 days was performed for dopamine denervation. The morphine, DAGO, DPDPE, and U5O,488H inhibited the increase of haloperidol-induced cyclic AMP content in chronic haloperidol treated mouse striatum. The inhibition of DAGO and DPDPE showed significant increase compared to normal mouse striatum. Naloxone showed antagonistic effect on the morphine and U5O,488H in chronic haloperidol treated group, and showed antagonistic effect on morphine, DAGO, DPDPE, and U5O, 488H in normal mouse striatum. These findings support that there is a functional interrelationship of dopaminergic and opioidergic pathway in the striatum. This result provides an evidence that following destruction of striatal dopaminergic neuron, there are some changes of cAMP content on the ${\mu},\;{\gamma},\;and\;{\kappa}$ opioid receptor, but the ${\kappa}$ opioid receptor still has its function.

• Reproductive and Developmental Biology
• /
• v.28 no.4
• /
• pp.211-219
• /
• 2004

The effects of adenosine 5'-triphosphate (ATP) and ATP analogs, P/sub 2y/ purinoceptor agonists, on growth of normal mouse mammary epithelial cells (NMuMG) were examined. Cells were plated onto 24 well plates in DMEM supplemented with 10 % fetal calf serum. After serum starvation for 24 hours, ATP, P/sub 2y/ purinoceptor agonists (AdoPP[NH]P, ATP-α-S, ATP-γ-S, β, γ-me-ATP and 2me-S-ATP), P/sub 2u/ purinoceptor agonist (UTP) and P/sub 2y/ purinoceptor antagonists (Reactive Blue 2, more selective to P/sub 2y/ receptor than PPADS; PPADS) were added. DNA synthesis was estimated as incorporation of 3H-thymidine into DNA (1 hour pulse with 1 μ Ci/ml, 18～19 hours after treatment). ATP, Adopp[NH]P, ATP-α-S or ATP-γ-S, significantly increased DNA synthesis at 1, 10 and 100 μM concentrations with dose-dependency (P<0.05), and the maximum responses of ATP and ATP analogs were shown at 100 μM concentration (P<0.05). The potency order of DNA synthesis was ATP≥ATP- γ -S>Adopp [NH]P>ATP-α-S. β, γ -me-ATP, 2me-S-ATP and UTP did not increase DNA synthesis. In autoradiographic analysis of percentage of S-phase cells, similar results were observed to those of DNA synthesis. Addition of 1, 10 or 100 μM Reactive Blue 2 or PPADS significantly decreased ATP (100 μM)-induced DNA synthesis, however, PPADS was less effective than Reactive Blue 2. In Elvax 40P implant experiment, ATP directly stimulated mammary endbud growth in situ suggesting the physiological regulator of ATP in mammary growth. ATP 100 μM rapidly increased MAPK activity, reaching a maximum at 5 min and then gradually decreasing to the base level in 30 min. ATP analogs, Adopp[NH]P and ATP-γ-S also increased MAPK activity, however, β, γ-me-ATP and 2me-S-ATP did not. The inhibitor of the upstream MAPK kinase (MEK), PD 98059 (25 μM), effectively reduced ATP (100 μM) or EGF(10 ng/ml, as positive control)-induced MAPK activity and DNA synthesis (P<0.05). These results indicate that ATP-induced DNA synthesis was prevented from the direct inhibition of MAPK kinase pathway. Overall results support the hypothesis that the stimulatory effects of normal mouse mammary epithelial growth by addition of ATP or ATP analogs are mediated through mammary tissue specific P/sub 2y/ purinoceptor subtype, and MAPK activation is necessary for the ATP-induced cell growth.

• Biomolecules & Therapeutics
• /
• v.7 no.2
• /
• pp.105-111
• /
• 1999

Nitric oxide (NO), a cellular messenger synthesized from L-arginine by NO synthase (NOS, EC.1.14.13.39), is considered to be a regulator of gastric secretion. In the present study, the role of NO in the regulation of exocrine secretion was investigated in rat gastric chief cells. Treatment of chief cells with carba-chol resulted in an increase in the arginine conversion to citrulline, the amount of $NO_{x}$, the release of pepsine-gen, and the level of cGMP Especially, carbachol-stimulated increase of arginine to citrulline transformation, the amount of $NO_{x}$, cGMP level and the release of pepsinogen were partially reduced by the natural NOS inhibitor, $N^{G}$-monomethyl-L-arginine (MMA) and $N^{G}$, $N^{G}$-dimethyl-L-arginine (DMA). Furthermore, MMA- and DMA-induced decrease of pepsinogen secretion showed dose-dependent patters. Activation of NOS is one of the early events in receptor-mediated cascade of reactions in gastric chief cells and NO, not completely, but partially mediates gastric secretion. Agonist-stimulated pepsinogen secretion in chief cells has been considered to be mediated in adenosine 3',5'-cyclic monophosphate pathway and/or guanosine 3', 5'-cyclic monophosphate (cGMP) pathway. Taken together, the above results suggest that partial decrease of exocrine secretion following treatment of NOS inhibitor may result from the inactivation of NOS and subsequent guano- late cyclase, and NO/cGMP pathway may play a pivotal role in exocrine secretion.

• Development and Reproduction
• /
• v.23 no.3
• /
• pp.223-229
• /
• 2019

Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance (IR). T2DM is correlated with obesity and most T2DM medications have been developed for enhancing insulin sensitivity. Silk protein fibroin (SPF) from spiders has been suggested as an attractive biomaterial for medical purposes. We generated transgenic rice (TR) expressing SPF and fed it to diabetic $BKS.Cg-m+/+Lepr^{db}$ mice to monitor the changes in blood glucose levels and adipose tissue proteins associated with energy metabolism and insulin signaling. In the present study, the adipocyte size in abdominal fat in TR-SPF-fed mice was remarkably smaller than that of the control. Whereas the adenosine monophosphate-activated protein kinase (AMPK)-activated protein kinase and insulin receptor substrate 1 (IRS1) protein levels were increased in abdominal adipose tissues after TR-SPF feeding, levels of six-transmembrane protein of prostate 2 (STAMP2) proteins decreased. Phosphorylation of AMPK at threonine 172 and IRS1 at serine 307 and tyrosine 632 were both increased in adipose tissues from TR-SPF-fed mice. Increased expression and phosphorylation of IRS1 at both serine 307 and tyrosine 632 in adipose tissues indicated that adipocytes obtained from abdominal fat in TR-SPF-fed mice were more susceptible to insulin signaling than that of the control. STAMP2 protein levels decreased in adipose tissues from TR-SPF-fed mice, indicating that STAMP2 proteins were reducing adipocytes that were undergoing lipolysis. Taken together, this study showed that TR-SPF was effective in reducing blood glucose levels in diabetic mice and that concurrent lipolysis in abdominal adipocytes was associated with alterations of AMPK, IRS1, and STAMP2. Increased IRS1 expression and its phosphorylation by TR-SFP were considered to be particularly important in the induction of lipolysis in adipocytes, as well as in reducing blood glucose levels in this animal model.

• Tuberculosis and Respiratory Diseases
• /
• v.62 no.4
• /
• pp.290-298
• /
• 2007

Background: The currently available diagnostic markers for pleural effusion have a limited role. The soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a molecule recently reported to play an important role in the myeloid cell mediated inflammatory response, and is up regulated in the body fluid by bacterial or fungal products. This study examined the expression of sTREM-1 in pleural effusion. Methods: Between April 2004 and December 2005, 48 patients with pleural effusions were enrolled in this study. The pleural fluids were taken and analyzed for the total protein, glucose, lactate dehydrogenase (LDH), adenosine deaminase (ADA), and sTREM-1. Bacterial cultures and cytology tests were also performed. Results: The clinical diagnoses were 17 parapneumonic, 14 tuberculous, and 13 malignant effusions. Four patients presented with transudates. The mean ages of the parapneumonic, tuberculous and malignant effusion groups were $57.1{\pm}19.7$, $49.5{\pm}18.6$, $66.9{\pm}15.5$, and $76.0{\pm}18.1$. respectively. The level of sTREM-1 expression was significantly higher in the parapneumonic effusions ($344.0{\pm}488.7$) than in the tuberculous effusions ($81.7{\pm}56.6$) and malignant effusions ($39.3{\pm}19.6$). With a cut-off value of 55.4pg/ml, the sensitivity and specificity for a parapneumonic effusion was 70.6% and 74.1%. Conclusion: sTREM-1 expression is significantly higher in parapneumonic effusions, suggesting its potential role as an additional diagnostic marker for pleural effusions.

• Asian-Australasian Journal of Animal Sciences
• /
• v.22 no.9
• /
• pp.1320-1327
• /
• 2009

This study examined the effects of nicotinamide on proliferation, differentiation, and energy metabolism in a primary culture of bovine adipocytes. After treatment of cells with 100-500 $\mu{M}$ nicotinamide, cell growth was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and cellular lipid content was assessed by Oil Red O staining and a triglyceride (TG) assay. Several factors related to energy metabolism, namely adenosine triphosphatase (ATPase) activity, nitric oxide (NO) content, nitric oxide synthase (NOS) activity, the number of mitochondria and the relative expression of glyceraldehydes-3-phosphate dehydrogenase (GAPDH), peroxisome proliferator-activated receptor-$\gamma$ ($PPAR_{\gamma}$) and inducible NOS (iNOS), were also investigated. Results showed that nicotinamide induced both proliferation and differentiation in bovine preadipocytes. Nicotinamide decreased NO production by inhibiting NOS activity and iNOS mRNA expression, and controlled lipolytic activity by increasing ATPase activity and the number of mitochondria. The present study provides further evidence of the effects of nicotinamide on lipid and energy metabolism, and suggests that nicotinamide may play an important role in the development of bovine adipose tissue in vivo. This emphasizes the importance of investigating bovine adipose tissue to improve our understanding of dairy cow physiology.

• Korean Journal of Biological Psychiatry
• /
• v.8 no.1
• /
• pp.3-19
• /
• 2001

Recent basic and clinical studies demonstrate a major role for neural plasticity in the etiology and treatment of depression and stress-related illness. The neural plasticity is reflected both in the birth of new cell in the adult brain(neurogenesis) and the death of genetically healthy cells(apoptosis) in the response to the individual's interaction with the environment. The neural plasticity includes adaptations of intracellular signal transduction pathway and gene expression, as well as alterations in neuronal morphology and cell survival. At the cellular level, repeated stress causes shortening and debranching of dendrite in the CA3 region of hippocampus and suppress neurogenesis of dentate gyrus granule neurons. At the molecular level, both form of structural remodeling appear to be mediated by glucocorticoid hormone working in concert with glutamate and N-methyl-D-aspartate(NMDA) receptor, along with transmitters such as serotonin and GABA-benzodiazepine system. In addition, the decreased expression and reduced level of brain-derived neurotrophic factor(BDNF) could contribute the atrophy and decreased function of stress-vulnerable hippocampal neurons. It is also suggested that atrophy and death of neurons in the hippocampus, as well as prefrontal cortex and possibly other regions, could contribute to the pathophysiology of depression. Antidepressant treatment could oppose these adverse cellular effects, which may be regarded as a loss of neural plasticity, by blocking or reversing the atrophy of hippocampal neurons and by increasing cell survival and function via up-regulation of cyclic adenosine monophosphate response element-binding proteins(CREB) and BDNF. In this article, the molecular and cellular mechanisms that underlie stress, depression, and action of antidepressant are precisely discussed.

• Journal of Periodontal and Implant Science
• /
• v.25 no.3
• /
• pp.635-647
• /
• 1995

Human polymorphonuclear leukocytes(PMN) constitute a first line of defense against all forms of injury and microbial challenge, which share a common cell lineage with macrophage. Microbial component LPS activates macrophages to produce IL-1, MIP-1${\alpha}$, -1${\beta}$, TNF-${\alpha}$ and IL-6, etc. Those cytokines have autocrine function to the macrophages, and paracrine function to other cell such as PMN and affect them to produce some biological functions. Having a responsive homogeneous cell line, HL-60, offers us the possibility of studying extensively on the function of PMN, which were not possible previously with peripheral PMN, due to the short-lived nature and difficulty of getting a purified PMN. In the present study, I performed MIP-1 receptor binding assay using HL-60 cell and human peripheral PMN. Also, in vitro antimicrobial assay was performed using differentiated or undifferentiated HL-60 cell. Differentiation was induced by treatment with 500 M of $N^6,O^2-dibutyryl$ adenosine 3'5' cyclic monophosphate(dbcAMP) (PMN-like cell), or 20ng/ml of 12-O-tetradecanoylphorbol-13-acetate(TPA) (macrophage/monocyte-like cell). Receptors for MIP-1${\alpha}$ were identified on dbcAMP-treated HL-60 as well as peripheral PMN. However, bound radioactive MIP-1${\alpha}$ on differentiated HL-60 was much higher than that of peripheral PMN, which suggest receptor number of differentiated HL-60 cell is higher than that of peripheral PMN. Although both of TPA and dbcAMP treatment significantly enhanced antimicrobial action of HL-60 cell, dbcAMP-treated cell(PMN-like HL-60) killed S.aureus more effectively in this experiment. TPA or dbcAMP treatment significantly enhanced antimicrobial action of undifferentiated HL-60 cell. MIP-1${\alpha}$ further increased enhancing effect of TPA or dbcAMP. IL-1${\alpha}$, however, increased only dbcAMP-induced enhancing effect of antimicrobial action of HL-60 cell. These results suggest that differentiated HL-60 cell could replace peripheral PMN in analysis of various biological functions of cytokines on PMN cell.