Ubiquitin sepcific protease 2 acts as a key modulator for the regulation of cell cycle by adiponectin and leptin in cancer cells

( Saroj Nepal ),( Anup Shrestha ),( Pil Hoon Park ) 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-

Adiponectin and leptin, both produced from adipose tissue, cause cell cycle arrest and progression, re-spectively in cancer cells. Ubiquitin specific protease-2 (USP-2). a deubiquitinating enzyme, is known to impair proteasorne-induced degradation of cyelin D1, a critical cell cycle regulator. Herein, we investi-gated the effects of these adipokines on USP-2 expression and its potential role in the modulation of cell cycle. Treatment with globular adiponectin (gAcrp) decreased, whereas leptin increased USP-2 expres-sion both in human hepatoma and breast cancer cells. In addition, overexpression or gene silencing of USP-2 affected cyelin D1 expression and cell cycle progression/arrest by adipokines. Adiponectin and leptin also modulated in vitro proteasomal activity, which was partially dependent on USP-2 expression. Taken together. our results reveal that modulation of USP-2 expression plays a crucial role in cell cycle regu-lation by adipokines. Thus. USP-2 would be a promising therapeutic target for the modulation of cancer cell growth by adipokines.

Modulation of Cell Death and Survival by Adipokines in the Liver.

Nepal, Saroj,Park, Pil-Hoon Pharmaceutical Society of Japan 2015 Biological & pharmaceutical bulletin Vol.38 No.7

<P>Adipokines, hormones predominantly produced from adipose tissue, have been shown to impart dynamic functions in the liver. Emerging evidence has shown that adipokines are also involved in modulating liver cell survival and/or death. Among the various adipokines, adiponectin and leptin directly regulate proliferation of hepatocytes, Kupffer cells, and hepatic stellate cells. Moreover, these adipokines control apoptosis and cell cycle of hepatic cancer cells in a complex manner. Adiponectin possesses both pro- and anti-proliferative properties, whereas leptin appears to play roles as a pro-survival hormone. Recent studies have revealed that regulation of cell death and proliferation is one of the critical factors regulating liver physiology by adipokines. In this review, we summarize the effects of adipokines on apoptosis and survival of liver cells and also demonstrate their implications in regulating various liver functions and decipher the underlying molecular mechanisms.</P>

Medicinal Chemistry : Regulatory Role of Autophagy in Globular Adiponectin-Induced Apoptosis in Cancer Cells

( Saroj Nepal ),( Pil Hoon Park ) 영남대학교 약품개발연구소 2015 영남대학교 약품개발연구소 연구업적집 Vol.25 No.-

Adiponectin,an adipokine predominantly secreted from adipose tissue, exhibits diverse biological responses,including metabo-lism of glucose and lipid, and apoptosis in cancer cells. Recently, adiponectin has been shown to modulate autophagy of cancer cells,the role of autophagy in apoptosis of cancer cell caused by adiponetin has not been explored. In the present study,we dem-onstrated that globular adiponectin(gAcrp)increase in caspae-3 activity,Bax,microtubule-associated protein light chain 3-ll(L C3 ll)protein levels,and dutoptposis in both HepG2 and MCF-7 cell lines,whereas induction of autophagy by rapamycin, an mTORinhibitor,significantly prevented gAcrp-induced Bax expression in HepG cells.These results implicate that induction of autophagy plays a regulatory role in adiponectin-induced apoptosis of cancer cells,and thus inhibition of autophagy would be a novel promising taiget to enhance the efficiercy of cancer cell apoptosis by adiponectin.

Globular adiponectin inhibits ethanol-induced apoptosis in HepG2 cells through heme oxygenase-1 induction

( Saroj Nepal ),( Mi Jin Kim ),( Amit Subedi ),( Eung Seok Lee ),( Chul Soon Yong ),( Jung Ae Kim ),( Wonku Kang ),( Mi Kyung Kwak ),( Dharamvie Singh Arya ),( Pil Hoon Park ) 영남대학교 약품개발연구소 2013 영남대학교 약품개발연구소 연구업적집 Vol.23 No.0

Hepatocellular apoptosis is an essential pathological feature of alcoholic liver disease. Adiponectin, an adipokine predominantly secreted from adipose tissue, has been shown to play beneficial roles in alcoholic liver disease against various inflammatory and pro-apoptotic molecules. However, the effects of adiponectin on ethanol-induced apoptosis in liver cells are largely unknown. Herein, we investigated the role of globularadiponectin (gAcrp) in the prevention of ethanol-induced apoptosis and further tried to decipher the potential mechanisms involved. In the present study, we demonstrated that gAcrp significantly inhibits both ethanol-induced increase in Fas ligand expression and activation of caspase-3 in human hepatoma cell lines (HepG2 cells), suggesting that gAcrp plays a protective role against ethanol-induced apoptosis in liver cells. This protective effect of gAcrp was mediated through adiponectin receptor R1 (adipoR1). Further, globular adiponectin treatment caused induction of heme oxygenase-1 (HO-1) through, at least in part, nuclear factor (erythroid-derived 2)-like 2, (Nrf2) signaling. Treatment with SnPP, a pharmacological inhibitor of HO-1, and knockdown of HO-1 with small interfering RNA (siRNA) restored caspase-3 activity suppressed by gAcrp, indicating a critical role of HO-1 in mediating the protective role of gAcrp in ethanol-induced apoptosis in liver cells. In addition, carbon monoxide, a byproduct obtained from the catabolism of free heme was found to contribute to the anti-apoptotic effect of adiponectin. In conclusion, these data demonstrated that globular adiponectin prevents ethanol-induced apoptosis in HepG2 cells via HO-1 induction and revealed a novel biological response of globular adiponectin in the protection of liver injury from alcohol consumption. ⓒ2012 Elsevier lnc. All rights reserved.

Activation of autophagy by globular adiponectin attenuates ethanol-induced apoptosis in HepG2 cells:Involvement of AMPK/FoxO3A axis

( Saroj Nepal ),( Pil Hoon Park ) 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Hepatocellular apoptosis is important pathological entity of alcoholic liver disease. Previously, we have shown that globular adiponectin (gAcrp) protects liver cells from ethanol-induced apoptosis by modulating an array of signaling pathways. In the present study, we investigated the role of autophagy induction by gAcrp in the suppression of ethanol-induced apoptosis and its potential mechanism(s) in liver cells. Here, we demonstrated that gAcrp significantly restores ethanol-induced suppression of autophagy-related genes,including Beclin-1 and microtuble-associated protein light chain (LC3B) both in primary rat hepato-cytes and human hepatoma cell line (HepG2). Globular adiponectin also restored autophagosome formation suppressed by ethanol treatment in HepG2. Furthermore, inhibitionof gAcrp-induced autophagic process by knock-down of LC3B prevented protection from ethanol-induced apoptosis. In particlar, the autophagic process induced by gAcrp was involved in the suppression of ethanol-induced activation of caspase-8 and expression of Bax. Moreover, knock-down of AMPK by small interfering RNA (siRAN) blocked gAcrp-induced expression of genes related to autophagy, which in turn prevented protection from ethanol-induced apoptosis, suggesting that AMPK plays an important role in the induction of autophagy and protection of liver cells by gAcrp. Finally, we also showed that gAcrp treatment induces translocation of the forkhead box O member protein FoxO3A, into the nucleus, which may play a role in the induction of autophagy-related genes. Taken together, our data demonstrated that gAcrp protects liver cells from ethanol-induced apoptosis via induction of autophagy. Further, the AMPK-FOXO3A axis plays a cardinal role in gAcrp-induced autophagy and subsequent inhibition of ethanol-induced apoptosis.

Modulation of Atg5 expression by globular adiponectin contributes to autophagy flux and suppression of ethanol-induced cell death in liver cells

( Saroj Nepal ),( Mi Jin Kim ),( Eung Seok Lee ),( Jung Ae Kim ),( Dong Yong Choi ),( Dong Hwan Sohn ),( Sung Hee Lee ),( Kyung Song ),( Sang Hyun Kim ),( Gil Saeng Jeong ),( Tae Cheon Jeong ),( Pil H 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Globular adiponectin (gAcrp) protects liver cells from ethanol-induced apoptosis via induction of autophagy. However, the underlying mechanisms are unknown. The present study aims to investigate the potential role of autophagy-related protein 5 (Atg5), an essential Atg for the elongation of autophagosomes, in suppression of ethanol-induced cytotoxicity by gAcrp. Here, we demonstrated that suppression of Atg5 expression by ethanol was restored by pretreatment with gAcrp both in primary rat hepatocytes and human hepatoma cell line (HepG2). Moreover, ethanol-induced accumulation of p62 (sequestosome1), a marker of autophagic flux, was restored by gAcrp treatment, implying that gAcrp modulates autophagic flux in liver cells. Further, Atg5 silencing prevented p62 degradation by gAcrp, suggesting that Atg5 plays a critical role in induction of autophagic flux by gAcrp. Interestingly, gene silencing of Atg5 by siRNA abrogated restoration of autophagosome formation by gAcrp in ethanol-treated cells. Finally, protection of liver cells by gAcrp from ethanol-induced apoptosis was also significantly attenuated by knocking-down of Atg5 expression, suggesting an important role of Atg5 in autophagy induction and cellular apoptosis modulated by gAcrp. Taken together, our data demonstrated that Atg5 expression, at least in part, is implicated in gAcrp-induced autophagy and subsequent anti-apoptotic effects in ethanol-treated liver cells.ⓒ2014Elsevier Ltd. All rights reserved

Regulatory Role of Autophagy in Globular Adiponectin-Induced Apoptosis in Cancer Cells

( Saroj Nepal ),( Pil Hoon Park ) 한국응용약물학회 2014 Biomolecules & Therapeutics(구 응용약물학회지) Vol.22 No.5

Adiponectin, an adipokine predominantly secreted from adipose tissue, exhibits diverse biological responses, including metabolism of glucose and lipid, and apoptosis in cancer cells. Recently, adiponectin has been shown to modulate autophagy as well. While emerging evidence has demonstrated that autophagy plays a role in the modulation of proliferation and apoptosis of cancer cells, the role of autophagy in apoptosis of cancer cell caused by adiponectin has not been explored. In the present study, we demonstrated that globular adiponectin (gAcrp) induces both apoptosis and autophagy in human hepatoma cell line (HepG2 cells) and breast cancer cells (MCF-7), as evidenced by increase in caspase-3 activity, Bax, microtubule-associated protein light chain 3-II (LC3 II) protein levels, and autophagosome formation. Interestingly, gene silencing of LC3B, an autophagy marker, significantly enhanced gAcrp-induced apoptosis in both HepG2 and MCF-7 cell lines, whereas induction of autophagy by rapamycin, an mTOR inhibitor, significantly prevented gAcrp-induced apoptosis in hepatoma cells HepG2. Furthermore, modulation of autophagy produced similar effects on gAcrp-induced Bax expression in HepG2 cells. These results implicate that induction of autophagy plays a regulatory role in adiponectin-induced apoptosis of cancer cells, and thus inhibition of autophagy would be a novel promising target to enhance the efficiency of cancer cell apoptosis by adiponectin.

Critical Role of AMPK/Fox03A Axis in Globular Adiponectin-induced Cell Cycle Arrest and Apoptosis in Cancer Cells

( Anup Shrestha ),( Saroj Nepal ),( Mijin Kim ),( Jae Hoon Chang ),( Sang Hyun Kim ),( Gil Saeng Jeong ),( Chul Ho Jeong ),( Gyu Hwan Park ),( Sunghee Jung ),( Jaecheong Lim ),( Eunha Cho ),( Soyoung 영남대학교 약품개발연구소 2016 영남대학교 약품개발연구소 연구업적집 Vol.26 No.-

Adiponeectin predominantly secreted from adipose tissue has exhibited potent anti- proliferative properties in cancer cells via modulating cell cycle and apoptosis, FoxO3A. a Forkhead box 0 member of the transcription factor. plays a critical role in modulating expression of genes involved In cell de:uh andlor SUrviV31. In this study. we investigated the role of FoxO3A signaling in and-cancer activities of adiponectin. Herein. we have shown that treaunent with globular adlponectln (gAcrp) Increases p27 but decreases cyclinD I expression In human hepatoma (HepG2) and breasr (MCF-7) cancer cells. Gene ablation of Fox03A prevented gAcrp-induced increase In p27 and decreased in cyelin 0 I expression. and further ameliorated cell cycle arrest by gAcrp.lndicating 3 critical role of FoxO3A in gAcrp-induced cell cycle arrest of cancer cells. Moreover, treatment with gAcrp also induced caspase-Jf7 activation and increased Fas ligand (Fasl) expression ln both HepG2 and MCF-7 cells. Transfecucn with FoxO3A siRNA inhibited gAcrp-induced caspase-3/7 activadon and Fasl expression. suggening that FoxO3A sigltaling also plays an lmportant role In gAcrp.induced apcptosis of cancer cells, We also found that gene silencing of AMPK prevented gAcrp-induced nuclear translocation of Fox03A in HepG2 and MCF-7 cells. In addition. suppression of AMPK also blocked gAcrp-lnduced cell cyele arrest and further attenuated gAcrp-induced caspase-3/7 activation, indICating that AMPK signaling plays a pivotal role in both gAcrp-lnduced cell cycle arrest and apoprosis via acting as an upstream S1gnalirlg of Fox03A Taken together. our firldings demonstrated that AMPK/Fox03A axis plays a cardinal role in anu-protlferanve effect of adiponecun in cancer cells.

Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

( Mi Jin Kim ),( Saroj Nepal ),( Eung Seok Lee ),( Tae Cheon Jeong ),( Sang Hyun Kim ),( Pil-hoon Park ) 영남대학교 약품개발연구소 2014 영남대학교 약품개발연구소 연구업적집 Vol.24 No.0

Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages.ⓒ2013 Elsevier lnc. All rights reserved.

Globular adiponectin modulates expression of programmed cell death 4 and miR-21 in RAW 264.7 macrophages through the MAPK/NK-kB pathway

( Amit Subedi ),( Mi Jin Kim ),( Saroj Nepal ),( Eung Seok Lee ),( Jung Ae Kim ),( Dong Hwan Sohn ),( Kyung Song ),( Sung Hee Lee ),( Won Sang Park ),( Byeong Seon Jeong ),( Pil Hoon Park ) 영남대학교 약품개발연구소 2013 영남대학교 약품개발연구소 연구업적집 Vol.23 No.0

MicroRNA-21 and programmed cell death 4 (PDCD4), a downstream target of miR-21, mediate diverse physiological responses. Here we demonstrate that globular adiponectin (gAcrp) modulates expressionof miR-21 and PDCD4 in RAW 264.7 macrophages. These effects were abrogated by inhibitors of ERK1/2, JNK or NF-κB. Conditioned media collected from gAcrp-stimulated RAW 264.7 macrophages caused similar effects as direct gAcrp treatment, showing the paracrine effect of gAcrp. These data indicate that gAcrp modulates the miR-21/PDCD4 axis through the ERK and JNK/NF-κB pathways in RAW 264.7 macrophages and further suggest that the miR-21/PDCD4 axis may be a novel target mediating adiponectin-induced biological responses.ⓒ2013 Federation of European Biochemical Societies. Published by Elsevier B.B. All rights reserved.