1.Sodium nitrite enhanced the potentials of migration and invasion of human hepatocellular carcinoma SMMC-7721 cells through induction of mitophagy.
Guan GUI ; Shan-shan MENG ; Lu-juan LI ; Bin LIU ; Hong-xia LIANG ; Chao-shen HUANGFU
Acta Pharmaceutica Sinica 2016;51(1):59-67
Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1 α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg x (-1) sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COX I and COXIV mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg x L(-1)) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.
Acetylcysteine
;
pharmacology
;
Autophagy
;
Carcinoma, Hepatocellular
;
pathology
;
Cell Line, Tumor
;
Cell Movement
;
Humans
;
Hypoxia-Inducible Factor 1, alpha Subunit
;
metabolism
;
Liver Neoplasms
;
pathology
;
Mitochondrial Degradation
;
Neoplasm Invasiveness
;
Nitrites
;
metabolism
;
Reactive Oxygen Species
;
metabolism
;
Sodium Nitrite
;
pharmacology
2.Sodium nitrite reduces lipid accumulation in steatotic cells by enhancing autophagy.
You-jing ZHANG ; Nai-rui ZHENG ; Bin LIU ; Ai-ling JI ; Yan-zhang LI ; Chao-shen HUANGFU
Acta Pharmaceutica Sinica 2015;50(8):1000-1007
Recent data have revealed that inhibiting autophagy exacerbates lipid accumulation in hepatocytes and nitrite treatment reduces total triglyceride levels in the high-fat diet mice. Therefore, the present study aimed to determine the effects of nitrite on simple hepatic steatosis and the possible role of autophagy. Firstly, steatotic L-02 cells were induced by incubating L-02 cells with 1.2 mmol · L(-1) oleic acid (OA) for 24 h. Secondly, steatotic L-02 cells were treated with 0.2 mmol · L(-1) sodium nitrite (SN) plus 3-methyladenine (3-MA), or chloroquine (CQ) for 24 h, and then lipid accumulation was measured with oil red O staining and triglyceride quantification. The notable steatosis could be observed in L-02 cells following exposure to 1.2 mmol · L(-1) OA for 24 h. Treatment with 0.2 mmol · L(-1) sodium nitrite reduced lipid accumulation in steatotic L-02 cells. 3-MA weakened the ability of sodium nitrite to ameliorate hepatic steatosis. Additionally, the sodium nitrite increased number of LC3-II immunostaining puncta and LC3-II protein expression was confirmed by immunofluorescence or Western blot analysis, and the effects were enhanced by CQ treatment. The number of increased cytoplasm vacuoles and lysosomes increased was confirmed by phase contrast and fluorescence microscope respectively. The increased autolysosome was detected by electron microscopy, this phenomenon could be reversed by CQ treatment. These data demonstrated that sodium nitrite enhanced the autophagic flux and decomposition of triglycerides in steatotic L-02 cells.
Adenine
;
analogs & derivatives
;
Autophagy
;
Blotting, Western
;
Cells, Cultured
;
Chloroquine
;
Cytoplasm
;
Fatty Liver
;
Hepatocytes
;
drug effects
;
Humans
;
Lipid Metabolism
;
drug effects
;
Microscopy, Fluorescence
;
Microtubule-Associated Proteins
;
metabolism
;
Oleic Acid
;
Sodium Nitrite
;
pharmacology
;
Triglycerides
3.Protective effects of sodium nitrite preconditioning against alcohol-induced acute liver injury in mice.
Xiao-Xia LU ; Si-Qian WANG ; Zhi ZHANG ; Hong-Rui XU ; Bin LIU ; Chao-Shen HUANGFU
Acta Physiologica Sinica 2012;64(3):313-320
The purpose of the present study was to investigate the effect of sodium nitrite (SN) on alcohol-induced acute liver injury in mice. Forty male C57bL/6 mice were randomly divided into 4 groups. Acute alcohol-induced liver injury group were injected intraperitoneal (ip) with alcohol (4.5 g/kg); SN preconditioning group were pretreated with SN (16 mg/kg, ip) for 12 h, and received alcohol (4.5 g/kg, ip) injection; Control and SN groups were treated with saline and SN, respectively. After the treatments, liver index (liver/body weight ratio) was determined. Colorimetric technique was performed to measure the serum alanine transaminase (ALT), aspartate transaminase (AST), liver superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) activities, as well as malondialdehyde (MDA) content. The pathological index of liver tissue was assayed by HE and TUNEL fluorometric staining. Using Western blot and immunohistochemistry staining, the expression of hypoxia-inducible factor-1α (HIF-1α) protein was detected. The results showed that, compared with acute alcohol-induced liver injury group, pretreatment with low doses of SN decreased liver index and serum levels of ALT and AST, weakened acute alcohol-induced hepatocyte necrosis, improved pathological changes in liver tissue, increased live tissue SOD, GSH-Px and CAT activities, reduced MDA content and apoptosis index of hepatocytes, and up-regulated HIF-1α protein level in liver tissue. These results suggest that the pretreatment of SN can protect hepatocytes against alcohol-induced acute injury, and the protective mechanism involves inhibition of oxidative stress and up-regulation of HIF-1α protein level.
Alanine Transaminase
;
metabolism
;
Alcohols
;
adverse effects
;
Animals
;
Apoptosis
;
Aspartate Aminotransferases
;
metabolism
;
Chemical and Drug Induced Liver Injury
;
drug therapy
;
Glutathione Peroxidase
;
metabolism
;
Hepatocytes
;
drug effects
;
Hypoxia-Inducible Factor 1, alpha Subunit
;
metabolism
;
Male
;
Malondialdehyde
;
metabolism
;
Mice
;
Mice, Inbred C57BL
;
Oxidative Stress
;
Protective Agents
;
pharmacology
;
Sodium Nitrite
;
pharmacology
;
Superoxide Dismutase
;
metabolism
;
Up-Regulation
4.Effect of exogenous hydrogen sulfide on lipophagy in mouse primary hep-atocytes
gang Hong WANG ; jing You ZHANG ; shen Chao HUANGFU ; liang Ming CHEN ; Jun WANG ; dong Dong WU ; xing Zhen XIE ; zhang Yan LI ; ling Ai JI
Chinese Journal of Pathophysiology 2017;33(10):1901-1905
AIM:To evaluate the effect of exogenous hydrogen sulfide ( H2 S) from GYY4137 on lipophagy in mouse primary hepatocytes .METHODS:The C57BL/6 mouse primary hepatocytes isolated and cultured by 2-step in situ perfusion method were divided into 4 groups:the cells in control group were incubated with normal medium; the cells in model group were incubated with 1.2 mmol/L oleic acid (OA) for 48 h;the cells in H2S group or propargylglycine (PAG) group were incubated with 1.2 mmol/L OA for 48 h followed by serum-free phenol red-free RPMI-1640 medium which con-tained 1 mmol/L GYY4137 or 200 μmol/L PAG for 6 h.The cells were collected to conduct immunofluorescence staining of LC3 and photography under fluorescence microscope , phase-contrast microscope or transmission electron microscope . The protein expression of LC 3-Ⅰ/Ⅱin the hepatocytes was determined by Western blot .RESULTS:In contrast with the model group, the fluorescent particles of LC3, the protein expression of LC3, the number of autophagic lysosome and vacuoles in hepatocytes in H 2 S group increased .CONCLUSION: In steatosis hepatocytes , exogenous H 2 S promotes the lipophagy .
5.Research progress of reactive nitrogen species on cell protection and injury
Su-juan CHEN ; Chen-chen PANG ; Bin LIU ; Chao-shen HUANGFU
Acta Pharmaceutica Sinica 2020;55(12):2785-2792
Reactive nitrogen species (RNS) affects intracellular redox balance and induces post-translational modification of proteins. Moreover, RNS, as the signal molecule, participates in the transduction of cellular signals under physiological conditions. However, excessive RNS can induce nitrosative stress and then damage cells, and thereby may play a role in the tumor initiation and progression. Thus, we discussed the role of RNS under physiological conditions and the tumor microenvironment, which may provide some novel ideas for the development of new drugs and the treatment of diseases.
6.Sodium nitrite enhanced the potentials of migration and invasion of human hepatocellular carcinoma SMMC-7721 cells through induction of mitophagy
Guan GUI ; Shan-shan MENG ; Lu-juan LI ; Bin LIU ; Hong-xia LIANG ; Chao-shen HUANGFU
Acta Pharmaceutica Sinica 2016;51(1):59-
Nitrites play multiple characteristic functions in invasion and metastasis of hepatic cancer cells, but the exact mechanism is not yet known. Cancer cells can maintain the malignant characteristics via clearance of excess mitochondria by mitophagy. The purpose of this article was to determine the roles of nitrite, reactive oxygen species (ROS) and hypoxia inducing factor 1 alpha (HIF-1α) in mitophagy of hepatic cancer cells. After exposure of human hepatocellular carcinoma SMMC-7721 cells to a serial concentrations of sodium nitrite for 24 h under normal oxygen, the maximal cell vitality was increased by 16 mg·L-1 sodium nitrite. In addition, the potentials of migration and invasion for SMMC-7721 cells were increased significantly at the same time. Furthermore, sodium nitrite exposure displayed an increase of stress fibers, lamellipodum and perinuclear mitochondrial distribution by cell staining with Actin-Tracker Green and Mito-Tracker Red, which was reversed by N-acetylcysteine (NAC, a reactive oxygen scavenger). DCFH-DA staining with fluorescent microscopy showed that the intracellular level of ROS concentration was increased by the sodium nitrite treatment. LC3 immunostaining and Western blot results showed that sodium nitrite enhanced cell autophagy flux. Under the transmission electron microscopy (TEM), more autolysosomes formed after sodium nitrite treatment and NAC could prevent autophagosome degradation. RT-PCR results indicated that the expression levels of COXⅠ and COXⅣ mRNA were decreased significantly after sodium nitrite treatment. Meanwhile, laser scanning confocal microscopy showed that sodium nitrite significantly reduced mitochondrial mass detected by Mito-Tracker Green staining. The expression levels of HIF-1α, Beclin-1 and Bnip3 (mitophagy marker molecular) increased remarkably after sodium nitrite treatment, which were reversed by NAC. Our results demonstrated that sodium nitrite (16 mg·L-1) increased the potentials of invasion and migration of hepatic cancer SMMC-7721 cells through induction of ROS and HIF-1α mediated mitophagy.
7.Exposure of human hepatoma cells to nitrite and ammonia promotes invasive activity through activation of ROS/ODC pathway
Shan-shan MENG ; Guan GUI ; Lu-juan LI ; Bin LIU ; Hong-xia LIANG ; Liang-ce LIU ; Chao-shen HUANGFU
Acta Pharmaceutica Sinica 2016;51(7):1083-
Recent studies have demonstrated that nitrite and ammonia levels are higher in the tumor environment, but their effects on cancer cells remains unclear. The present study was designed to determine the effects of nitrite and ammonia on tumor invasion and the role of reactive oxygen (ROS)/ornithine decarboxylase (ODC) pathway. SMMC-7721 cells were treated with sodium nitrite, ammonium chloride, sodium nitrite and ammonium chloride mixture for 24 h, the cell viability was analyzed using the MTT assay, cell invasion was analyzed with the transwell assay, the intracellular ROS levels were detected with a reactive oxygen species (ROS) test kits, the expression of intracellular ODC was examined with immunofluorescence and Western blot, the expression of matrix metallopeptidase-2(MMP-2) and MMP-9 were analyzed by Western blot. Compared with the control group, SMMC-7721 cells exhibited an increase in cell viability, invasion ability, ROS levels and ODC protein after exposure to 150 μmol·L-1 sodium nitrite and ammonium chloride mixture for 24 h. The invasive activity was reduced by ROS scavenger N-acetycysteine (NAC) in SMMC-7721 cells. The specific ODC inhibitor difluoromethylornithine (DFMO) increased ROS levels and weakened the ability of sodium nitrite and ammonium chloride mixture in the regulation of invasion of SMMC-7721 cells. These data demonstrated that sodium nitrite and ammonium chloride mixture promote invasion of SMMC-7721 cells by enhancing ROS/ODC pathway.
8.Involvement of sodium nitrite in the regulation of cancer stem cell properties in human hepatoma cells
Liang-ce LIU ; Shan-shan MENG ; Guan GUI ; Lu-juan LI ; Bin LIU ; Hong-xia LIANG ; Chao-shen HUANGFU
Acta Pharmaceutica Sinica 2017;52(8):1241-1249
Increasing evidence suggests that hepatocellular carcinomas (HCCs) are sustained by a distinct subpopulation of self-renewing cells known as cancer stem cells (CSC). However, our understanding of their regulation is limited. Rapid reversible changes of CSC-like cells within tumors may result from the effect of biological mediators found in the tumor microenvironment. This paper aims to explore how nitrite, a key cellular modulator whose level is elevated in many tumors, affects CSC-like phenotypes of human hepatoma cells SMMC-7721 cells. The SMMC-7721 cell line was cultured under serum-free conditions to produce floating spheres. The distribution of cell cycle was analyzed by flow cytometry, the capability of cells self-renew was detected by colony-forming capabilities and spheroid-formation assay, the expression of stemness protein such as CD133, CD90 and EpCAM were determined by flow cytometry and Western blot, cell invasion was analyzed by transwell assay, and viability of SMMC-7721 parental cells and spheroids cancer cells was determined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Xenograft tumor models were established by subcutaneously injecting SMMC-7721 spheroids cancer cells, the transplanted tumor tissue ROS levels was detected by reactive oxygen species (ROS) test kits, the expression of HIF-1α was observed by immunofluorescence. Our results showed that the SMMC-7721 spheroid cells were enriched with CSCs properties, indicated by the ability to self-renew, increased expression of CSCs markers, and increased resistance to chemotherapeutic drugs. Additionally, SMMC-7721 parental cells and spheroids cancer cells were treated with 150 μmol·L-1 sodium nitrite for 6 days, compared with control cells, an increased accumulation of G0/G1 phase cells was observable in treatment cells. Indeed, our data demonstrated that in parent cells and spheres cells that were treated with sodium nitrite for different time, the cells' ability to chemoresistance and invasion, clone-forming efficiencies and the spheres forming ability were significantly higher than that of control cells. Exposure of sodium nitrite regulated CSC-like phenotype, indicated by increased expression of known CSC markers, CD133, CD90 and EpCAM in the exposed parental cells, as well as in dormant spheroids cancer cells. Compared with the parent cells, the above effects of nitrite on the spheres cells were significantly enhanced. In vivo data also presented a more significant promotion of tumor xenograft growth from the nitrite treatment than from either of the control. Mechanistic analysis indicated that nitrite induced the upregulation of HIF-1α as well as the downregulation of ROS in the tumor microenvironment. These results suggest that nitrite increases the invasiveness of SMMC-7721 cells through up-regulation of tumor stemness.
9.A new stage for healthy China: new characteristics and urgency of public health
Mo HAO ; Peiwu SHI ; Qunhong SHEN ; Zhaoyang ZHANG ; Zheng CHEN ; Chuan PU ; Lingzhong XU ; Zhi HU ; Anning MA ; Zhaohui GONG ; Tianqiang XU ; Panshi WANG ; Hua WANG ; Chao HAO ; Huihui HUANGFU ; Yang CHEN ; Qingyu ZHOU ; Chengyue LI
Shanghai Journal of Preventive Medicine 2022;34(1):22-27
Building a strong public health system has become an urgent task in the new era. Based on more than eight years of systematic research, we believe that five aspects need to be prioritized for a strong system. First, we should change the perspective on public health, using the word “gonggong jiankang” to replace “gonggong weisheng” and the word “gonggong jiankang tixi” to replace “gonggong weisheng tixi”, to lead the public health system development. Second, we should develop a suitable public health system and continuously improve the health capacity for governance. Third, we should make it clear that the goal of building a strong system is not far-fetched, and we need to consolidate the existing institutional advantages of China’s public health system: when encountering major problems, we can maintain a unified goal and mobilize the whole society to cooperate effectively to accomplish the goal. However, we need to make up for shortcomings one by one, especially to solve the key problem of lacking a strong coordination mechanism in daily work. Fourth, we should pursue excellence and consolidate the“suitable” mechanism proven in the process of coping with the COVID-19, so that efficient mechanisms to deal with major issues can be used in routine work, and efforts should be made to consolidate the advantages of prevention and control of infectious diseases and emergency response,so as to achieve the balanced development of regions categories and units.Finally, it is necessary to strengthen the coordination of government and research institutions, in the aspects of technological innovation, talent team building and accurate consulting services, and work together to pursue a suitable and strong system to realize the modernization of the health system and capacity for governance.