Inhibitory Effect of Sesquiterpenoid M36 from Myrrha on Growth of Human Hepatoma Cells / 中国实验方剂学杂志

ObjectiveThe antitumor activity of sesquiterpenoid M36 isolated from Myrrha against human hepatoma HepG2 cells was investigated in this study. MethodHepG2 cells were treated with M36 at different concentrations （0， 2， 4， 6， 8， 10 μmol·L-1）. Firstly， the effects of M36 on the proliferation of human hepatoma HepG2 cells were detected by methyl thiazolyl tetrazolium （MTT）， colony formation assay， and EdU proliferation assay. Hoechst staining， flow cytometry analysis， and Western blot were used to explore the effect of M36 on the apoptosis of human hepatoma HepG2 cells. Acridine orange staining and western blotting were used to examine the effect of M36 on autophagy in HepG2 cells. Finally， Western blot was used to detect protein expression of cancer-related signaling pathways. ResultCompared with the blank group， M36 treatment significantly inhibited the proliferation of human hepatoma HepG2 cells （P<0.01）， and the half inhibitory concentration （IC50） value of M36 for 48 h was 5.03 μmol·L-1， in a dose- and time-dependent manner. M36 was also able to induce apoptosis and autophagy in human hepatoma HepG2 cells. After treatment with 8 μmol·L-1 M36 for 48 hours， the apoptosis rate of HepG2 cells was （42.03±9.65）% （P<0.01）. Compared with the blank group， HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h had a significant increase in cleaved poly ADP-ribose polymerase （cleaved-PARP） protein levels （P<0.01）. Acridine orange staining showed that autophagy was significantly activated in HepG2 cells treated with 4 and 8 μmol·L-1 M36 for 48 h compared with the blank group （P<0.01）， which was further verified by the up-regulation of microtubule-associated protein 1 light chain 3 Ⅱ （LC3 Ⅱ）. Western blot results showed that compared with the blank group， the levels of phosphorylated extracellular regulated protein kinase （p-ERK）， phosphorylated p38 mitogen-activated protein kinase （p-p38 MAPK）， phosphorylated c-Jun N-terminal kinase （p-JNK）， and its downstream nuclear transcription factors c-Jun and p-c-Jun protein were significantly increased in M36 group （P<0.05， P<0.01）. The mechanism may be related to the up-regulation of MAPK signaling pathway. ConclusionThe sesquiterpenoid M36 isolated from Myrrha inhibits the proliferation of human hepatoma HepG2 cells and promotes apoptosis and autophagy， which may be related to the activation of the MAPK signaling pathway.

Mechanism of Proteoglycan TPG-1 from Trametes robiniophila Inhibiting Growth of Human Hepatoma SK-HEP-1 Cells / 中国实验方剂学杂志

ObjectiveProteoglycan TPG-1 isolated from Trametes robiniophila（Huaier） has proved to have anti-hepatoma activity， and this paper aims to explore the molecular mechanism. MethodHuman hepatoma SK-HEP-1 cells were treated with TPG-1 （0， 0.05， 0.1， 0.25， 0.5， 1 g·L-1）. Then cell survival was detected by methyl thiazolyl tetrazolium （MTT） and apoptosis by flow cytometry. In addition， expression of genes in SK-HEP-1 cells treated with or without TPG-1 was examined by DNA microarray to preliminarily explore the anti-hepatoma molecular mechanism of TPG-1. ResultTPG-1 inhibited the proliferation of SK-HEP-1 cells as compared with the blank group （P<0.01）. After treatment with 1 g·L-1 TPG-1 for 48 h， the apoptosis rate of SK-HEP-1 cells increased （P<0.01）， and TPG-1 promoted the cleavage of cysteinyl aspartate specific proteinase （Caspase）-3 and Caspase-7， the key mediators of apoptosis （P<0.01）. Additionally， TPG-1 （1 g·L-1） suppressed the migration of SK-HEP-1 cells （P<0.05）. A total of 971 differentially expressed genes （DEGs） were identified in SK-HEP-1 cells after treatment with TPG-1， with 486 up-regulated and 485 down-regulated. These DEGs were mainly involved in the Gene Ontology （GO） terms of interleukin-6 （IL-6） biosynthesis， antigen processing and presentation， superoxide dismutase activity， positive regulation of mitogen-activated protein kinase kinase kinase （MAPKKK） cascade， nature killer （NK） cell chemotaxis， and chemokine biosynthesis， and the Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways of nucleotide-binding oligomerization domain （NOD）-like receptor signaling pathway， apoptosis， Toll-like receptor signaling pathway， retinoic acid-inducible gene-Ⅰ （RIG-Ⅰ）-like receptor signaling pathway， T-cell receptor signaling pathway， and chemokine signaling pathway. Western blot results showed that TPG-1 （1 g·L-1） activated mitogen-activated protein kinase （MAPK） signaling pathway in SK-HEP-1 cells （P<0.01）. ConclusionProteoglycan TPG-1 inhibited the proliferation and migration， and induced apoptosis of human hepatoma SK-HEP-1 cells. Up-regulation of MAPK signaling pathway may be responsible for the growth inhibition of human hepatoma SK-HEP-1 cells by TPG-1.

Effect of Stemona tuberosa Alkaloids on Apoptosis of SMMC-7721 Cells and Expression of Bcl-2， Bax， and Cleaved Caspase-3 / 中国实验方剂学杂志

Objective:To investigate the effect of<italic> Stemona tuberosa</italic> alkaloids on the apoptosis of human hepatoma SMMC-7721 cells and the expression of apoptosis-related proteins including B lymphocytoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and cleaved cysteinyl aspartate-specific protease-3 （cleaved Caspase-3）. Method:SMMC-7721 cells were routinely cultured， passaged， and treated with various concentrations （50， 75， 112， 167， and 250 mg·L^-1） of <italic>S. tuberosa </italic>alkaloids， while those in the blank control group were only treated with 10% fetal bovine serum. The cell proliferation was determined by tetrazolium bromide （MTT） colorimetry and colony assay and the cell apoptosis by Hoechst 33258 staining. The protein expression levels of Bcl-2， Bax， and cleaved Caspase-3 were detected by Western blot. Result:<italic>S. tuberosa</italic> alkaloids inhibited the proliferation of SMMC-7721 cells， and the inhibition rate was significantly increased in comparison with that in the blank control group （<italic>P</italic><0.01）， with the half maximal inhibitory concentrations （IC₅₀） at 24 h， 48 h， and 72 h being （173.36±8.75）， （112.14±16.50）， and （96.41±2.60）mg·L^-1， respectively. The cell colony-inhibitory activity was significantly increased in a dose-dependent manner （<italic>P</italic><0.01）. Compared with the blank control group， <italic>S. tuberosa</italic> alkaloids promoted the apoptosis of SMMC-7721 cells， manifested as increased number of apoptotic cells and elevated apoptotic rate （<italic>P</italic><0.01）. The typical morphological changes such as brightly blue-fluorescent condensed nuclei， cytoplasmic shrinking， and karyopyknosis were found under the upright fluorescence microscope. As revealed by comparison with the blank control group， the expression of Bcl-2 was significantly down-regulated （<italic>P</italic><0.01）， while the protein expression levels of pro-apoptotic protein Bax and cleaved Caspase-3 in the 75， 112， 167， and 250 mg·L^-1 <italic>S. tuberosa</italic> alkaloids groups were significantly up-regulated （<italic>P</italic><0.01）. Conclusion:<italic>S. tuberosa </italic>alkaloids inhibit the proliferation of SMMC-7721 cells and promote their apoptosis possibly by inhibiting Bcl-2 protein expression and promoting Bax and cleaved Caspase-3 protein expression.

Improvement Effects of α-lipoic Acid on Glucose Metabolism Disorder of Insulin Resistant HepG 2 Cells / 中国药房

OBJECTIVE：To study the i mprovement effects of α-lipoic acid on glucose metabolism disorder of insulin resistant HepG2 cells. METHODS ：The effects of 25-1 000 µmol/L α-lipoic acid on survival rate of human hepatoma cell HepG2 were determined by MTT assay so as to determine the concentration of α-lipoic acid. Negative control group ，insulin resistance group （1× 10－7 mol/L insulin ），combination resistance group （30 µmol/L sodium arsenite+ 1×10－8 mol/L insulin ），α-lipoic acid low- concentration，medium-concentration and high-concentration groups were set up. HepG 2 cells were treated with α-lipoic acid for 12 h and then cultured with corresponding concentration of sodium arsenite or/and insulin for 24 h. The glucose oxidase method was used to detect the glucose consumption ，colorimetric method was used to detect hexokinase activity and pyruvate kinase activity ， and anthrone method was used to detect glycogen content. Western blot assay was used to detect the protein expression of GLUT 4， p-GSK3β and GSK3β as well as the ratio of p-Akt/Akt and p-GSK3β/GSK3β. RESULTS：25，50，100 µmol/L α-lipoic acid had no significant effect on the survival rates of HepG 2 cells（P＞0.05），and survival rates of H epG2 cells were higher than 96％，so they were used as the low ，medium and high concentration for follow-up study. Compared with negative control group ，glucose consumption，the activities of hexokinase and pyruvate kinase ，glycogen content ，protein expression of GLUT 4 and p-GSK 3β，the ratio of p-Akt/Akt and p-GSK 3β/GSK3β were decreased significantly in insulin resistance group and combined resistance group， while the protein expression of GSK 3β was increased significantly（P＜0.05）. Compared with combination resistance group ，the glucose consumption （except for α-lipoic acid low- concentration group ），the activities of h exokinase（except for α-lipoic acid low-concentration and medium-concentration groups ） andpyruvate kinase （except for α-lipoic acid low-concentration com and medium-concentration groups ）， glycogen contents ， protein expression of GLUT 4 （except for α-lipoic acid mail：bliang163@163.com low-concentration group ）and p-GSK3β，the ratio of p-Akt/ Akt（except for α-lipoic acid low-concentration and medium-concentration groups ）and p-GSK 3β/GSK3β（except for α-lipoic acid low-concentration groups ）were increased significantly in α-lipoic acid groups ，while protein expression of GSK 3β（except for α-lipoic acid low-concentration and medium-concentration groups ） was decreased significantly （P＜0.05）；glycogen content ， protein expression of GLUT 4 and the ratio of p-GSK 3β/GSK3β in α-lipoic acid high-concentration group as well as the protein expression of p-GSK 3β in α-lipoic acid medium-concentration and high-concentration groups were improved significantly （P＜0.05）. CONCLUSIONS：α-lipoic acid can improve the disorder of glucose metabolism in insulin resistant HepG 2 cells，the mechanism of which may be associated with the increase of glucose consumption ，the activities of glucose metabolism related enzymes and glycogen content ，and expression up-regulation of the phosphorylation levels of Akt and GSK 3β protein，the expression of GLUT 4 and p-GSK 3β proteins，down-regulation of the expression of GSK 3β protein.

Screening of optimal siRNA interference sequence of CIT gene and its inhibition expression in hepatoma SK-Hep-1 / 中华肝脏病杂志

Objective@#To construct and screen optimal siRNA interference sequence of CIT gene and to detect its interference efficiency as well as proliferation effect in human hepatoma cell line SK-Hep-1.@*Methods@#Three siRNA target spots were designed and synthesized according to the CIT gene sequence. SK-Hep-1 HCC cells were transfected by liposome transfection. The knockdown efficiency of the target CIT gene was detected by real-time PCR and Western blot. Expressional change of CIT in SK-Hep-1 cells after 48 hours of siRNA interference were observed by immunohistochemistry and confocal microscopy. The proliferation of SK-Hep-1 cells after 48 hours of siRNA interference was detected by EdU cell proliferation assay. A t-test was used to compare the mean of two samples, and one-way ANOVA was used to compare the mean of multiple samples.@*Results@#Western blot results showed that the three interference sequences were targeted at different target spots. The expression level of CIT protein in KD-1,-2, and-3 groups were decreased (P < 0.01) than control, while the protein expression level of KD1 group was the lowest. Real-time PCR results showed that compared with the control group, the expression level of CIT mRNA in KD-1, -2, and -3 groups decreased (P < 0.01), while that in KD1 group was the lowest. Laser confocal microscopy also confirmed that the morphological expression of CIT attenuated significantly after transfection with siRNA. The results of EdU proliferation assay showed that siRNA transfected with CIT significantly attenuated the proliferation of SK-Hep-1 hepatoma cells (P < 0.05).@*Conclusion@#The successful construction and screening of siRNA fragments can effectively inhibit the expression and proliferation of CIT gene in hepatoma SK-Hep-1.

Study on Synthesis and Anti-tumor Activity of 6 Kinds of Tectorigenin Mannich Base Derivatives / 中国药房

OBJECTIVE： To conduct structural modification of tectorigenin to search for new compounds with anti-tumor activity. METHODS： Tectorigenin was used as a lead compound， and then added into amine reagents as ethanolamine， methylamine， ethylamine， dimethylamine， diethylamine， n-propylamine and formaldehyde solution. Tectorigenin Mannich base derivatives were synthesized by mannich reaction with as the lead compound. The structures of the derivatives were identified according to IR， UV， MS and NMR data. Solubility of tectorigenin and its derivatives were investigated by solubility test method. MTT assay was used to investigate the inhibitory effects of tectorigenin and its derivatives on the proliferation of human colon cancer cell line HCT116， human lung cancer cell line A549 and human hepatoma cell line HepG2， and half inhibitory concentration （IC50） was calculated. The inhibition rate of tectorigenin and its derivatives （100 mg/kg） on H22 hepatoma-bearing mice in vivo was studied. RESULTS： Totally of 6 kinds of tectorigenin mannich base derivatives were synthesized， such as 8-（N-hydroxyethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-methyl）-methyleneamino-5，7，4′-trihydroxy-6- methoxyisoflavone， 8-（N， N-diethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N， N-dimethyl）-methyleneamino- 5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-ethyl）-methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone， 8-（N-propyl）- methyleneamino-5，7，4′-trihydroxy-6-methoxyisoflavone （compounds 1-6 in turn）. Compared with tectorigenin， the water solubility of six derivatives was significantly improved， and the solubility was 5-20 times higher than that of tectorigenin. IC50 of compounds 1， 3 and 5 to HCT116 cells were （34.82±3.27）， （16.21±4.13）， （33.12±3.25） μmol/L， which were stronger than that of tectorigenin [（45.23±5.74） μmol/L]； IC50 of compounds 1， 3 and 5 to A549 cells were （37.05±5.74）， （26.88±4.52）， （30.13±6.23） μmol/L， which were stronger than that of tectorigenin [（53.24±6.34） μmol/L]； IC50 of compounds 1， 3 and 5 to HepG2 cells were （23.74±1.45）， （18.96±2.34）， （30.95±2.87） μmol/L， which were stronger than that of tectorigenin [（48.98±2.58） μmol/L]. Compounds 1， 3 and 5 showed higher inhibition rates （55.51％， 57.20％ and 49.15％） than tectorigenin （33.05％） on H22 hepatoma-bearing mice， respectively. The other three compounds had no obvious advantage over tectorigenin in anti-tumor activity. CONCLUSIONS： In this study， compounds 1， 3 and 5 of six tectorigenin mannich base derivatives synthesized in this study have stronger antitumor activity than tectorigenin.

Characterization of In vitro inhibitory effects of consensus short interference RNAs against non-structural 5B gene of hepatitis C virus 1a genotype

Purpose: Chronic hepatitis C has infected approximately 170 million people worldwide. The novel direct-acting antivirals have proven their clinical efficacy to treat hepatitis C infection but still very expensive and beyond the financial range of most infected patients in low income and even resource replete nations. This study was conducted to establish an in vitro stable human hepatoma 7 (Huh-7) cell culture system with consistent expression of the non-structural 5B (NS5B) protein of hepatitis C virus (HCV) 1a genotype and to explore inhibitory effects of sequence-specific short interference RNA (siRNA) targeting NS5B in stable cell clones, and against viral replication in serum-inoculated Huh-7 cells. Materials and Methods: In vitro stable Huh-7 cells with persistent expression of NS5B protein was produced under gentamycin (G418) selection. siRNAs inhibitory effects were determined by analysing NS5B expression at mRNA and protein level through reverse transcription-polymerase chain reaction (PCR), quantitative real-time PCR, and Western blot, respectively. Statistical significance of data (NS5B gene suppression) was performed using SPSS software (version 16.0, SPSS Inc.). Results: siRNAs directed against NS5B gene significantly decreased NS5B expression at mRNA and protein levels in stable Huh-7 cells, and a vivid decrease in viral replication was also exhibited in serum-infected Huh-7 cells. Conclusions: Stable Huh-7 cells persistently expressing NS5B protein should be helpful for molecular pathogenesis of HCV infection and development of anti-HCV drug screening assays. The siRNA was effective against NS5B and could be considered as an adjuvant therapy along with other promising anti-HCV regimens.

Apoptosis of Human Hepatoma Cell Line SMMC-7721 Induced by Dihydroartemisinin and Its Possible Mechanism / 中国药学杂志

OBJECTIVE: To investigate the apoptosis effect of human hepatocellular carcinoma cell line SMMC-7721 induced by dihydroartemisinin in vitro and the possible mechanism. METHODS: After treatment with 25, 50, 100, 200, and 400 μmol•L-1 dihydroartemisinin for 24 h. The proliferation inhibitory effect of dihydroartemisinin on SMMC-7721 cell was detected by MTT assay. Cell cycle and apoptosis were detected by flow cytometry. The change of apoptotic morphology was detected by confocal laser scanning microscopy. Rho 123 staining method was used to detect the changes of mitochondrial membrane potential. Western blot was used to detect expression of Bcl-2, Bax, Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C. RESULTS: MTT results showed that 25-400 μmol•L -1 dihydroartemisinin can inhibit the proliferation of SMMC-7721 cells obviously. The cell cycle detection results of flow cytometry showed that dihydroartemisinin could block SMMC-7721 cell cycle in G2/M phase. The results of Hochest 333258 staining showed that the nuclei were heterogeneous, condensed and fragmented in the DHA treatment group. The cell apoptosis detection results of flow cytometry showed that the apoptosis rate of dihydroartemisinin treated groups were increased obviously (P < 0.01). The results of Rho 123 staining showed that the mitochondrial membrane potential was decreased significantly (P < 0.01). Western blot results showed that the expression of Bcl-2 was down-regulated, expression of Bax was up-regulated, the ration of Bax /Bcl-2 was increased and the expression of Cleaved Caspase-3, Cleaved Caspase-9 and Cyto C were up-regulated. CONCLUSION: Dihydroartemisinin can induce apoptosis of SMMC-7721 cells, on the mechanism of apoptosis may be related to mitochondrial pathway.

Huaier aqueous extract inhibits proliferation of human hepatoma SK-HEP-1 cells through up-regulation of autophagy / 中国中药杂志

The purpose of this study was to investigate the effect of Huaier on autophagy of human hepatoma SK-HEP-1 cells and the effect of autophagy on the proliferation of SK-HEP-1 cells. CCK-8 assay was used to evaluate the effect of Huaier on the proliferation of SK-HEP-1 cells under different concentrations and different times. Acridine orange staining was used to measure the effect of Huaier on the autolysosome formation in SK-HEP-1 cells. Immunofluorescence assay was applied to examine the effect of Huaier on the expression and distribution of autophagy marker LC3 in SK-HEP-1 cells. In addition， LC3 expression was also checked by immunoblot analysis in the presence of Huaier. At last， the effects of Huaier in combination with autophagy inhibitor bafilomycin A1 on the proliferation of SK-HEP-1 cells was detected by CCK-8 assay. The results showed that Huaier aqueous extract significantly inhibited the proliferation of human hepatoma SK-HEP-1 cells in a dose- and time-dependent manner. Huaier aqueous extract dramatically promoted the formation of autolysosome in SK-HEP-1 cells. Moreover， Huaier markedly increased the number and intensity of intracellular LC3 fluorescent puncta and up-regulated LC3-Ⅱ expression. These data indicated that Huaier evidently activated autophagy of SK-HEP-1 cells. Additionally， autophagy inhibition significantly attenuated the sensitivity of SK-HEP-1 cells to Huaier treatment. Therefore， autophagy activation is involved in the inhibitory effects of Huaier on the proliferation of human hepatoma SK-HEP-1 cells.

Effects of Betulinic Acid on Proliferation of Human Liver Cancer HepG2 Cells / 中国中医药信息杂志

Objective To observe the effects of betulinic acid (BA) on proliferation of human hepatoma stem cell;To discuss its anti-cancer mechanism from the aspects of cell cycle and cell apoptosis. Methods HepG2 stem cells were cultivated in vitro and testified the self-renewal capacity. The effects of BA in concentration of 40, 20, 10, 5, 2.5, 1.25μmol/L on the cell vitality of cultured human liver cancer stem cells for 24 and 48 hours were measured with CCK-8 method. The human hepatoma stem cell line HepG2 was administrated by BA at concentrations of 40, 20, 10, 5μmol/L for 48 hours, and cell cycle and apoptosis rate were measured by flow cytometry. Results BA could inhibit HepG2 stem cell proliferation obviously with dose-effect relationship. BA influenced cell cycle, and induced tumor stem cell apoptosis. 40μmol/L BA blocked cell cycle in S phase, and cell apoptosis rate reached 10.86%. Conclusion BA has obvious inhibitory effects on proliferation of HepG2 liver cancer stem cell, which probably plays a part in anti-cancer by influencing cell cycle and inducing cell apoptosis.

Study on the Effects and Mechanism of Celastrol on Proliferation and Apoptosis of Human Hepatoma HepG2 Cells in vitro / 中国药房

OBJECTIVE:To study the effects of celastrol on the proliferation and apoptosis of human hepatoma HepG2 cells, and investigate its mechanism. METHODS:CCK-8 method was used to determine the cell activity 24,48,72 h after treated by 2, 5,10 μmol/L celastrol,and the proliferation inhibition rate and half inhibitory concentration(IC50)were calculated;flow cytome-try was conducted to detect the cell apoptosis rate and cycle change 24 h after treated by 2,5,10μmol/L celastrol,and the DMSO was used as negative control;rhodamine 123 staining method was used to determine the mitochondrial membrane potential 48 h af-ter treated by 2,5,10 μmol/L celastrol,and the DMSO was used as negative control;Western blot was adopted to detect the pro-apoptotic related genes Bax and B lymphoma 2(Bcl-2)protein expressions 0,12,24,36 h after treated by 5 μmol/L celastrol. RESULTS:2,5,10 μmol/L celastrol can inhibit cell proliferation,IC50 was 5.834 μmol/L. 2,5,10 μmol/L celastrol can induce apoptosis;5,10 μmol/L celastrol can block cell in G0/G1,S phases,compared with negative control group,with significant differ-ences(P<0.05 or P<0.01),and the above effects all showing certain concentration-dependent manner. 5 μmol/L celastrol can in-crease Bax protein expression and decrease Bcl-2 protein expression after cultured for 12,24,36 h,showing certain time-depen-dent manner;compared with 0 h,there was significant difference(P<0.05 or P<0.01). CONCLUSIONS:Celastrol can obvious-ly inhibit the proliferation of human hepatoma HepG2 cells and induce their apoptosis,and the mechanism may be related with strengthening mitochondrial permeability and promoting the release of apoptosis-inducing factor.

Preparation of TPGS-modified artesunate liposomes and their in vitro anti-tumor activity / 中成药

AIM To prepare D-α-tocopherol polyethylene glycol 1000 succinate (TPGS)-modified artesunate liposomes and to investigate the in vitro anti-tumor activity.METHODS The liposomes prepared by thin-film dispersion method were characterized by transmission electron microscopy and particle size analyzer,and the encapsulation efficiency was determined by ultrafiltration centrifugation.The liposomes' cytotoxicity to human hepatoma HepG2 cells was evaluated by MTT method.RESULTS The average particle size,PDI,Zeta potential,encapsulation efficiency,drug loading of the liposomes were 126.7 nm,0.182,-10.1 mV,78.8％ and 18.38％,respectively.The liposomes displayed a significant inhibition on HepG2 cells with the IC50 value of 0.034 μmol/mL.CONCLUSION Compared with non-TPGS-modified artesunate liposomes,the TPGS-modified artesunate liposomes prepared by this method afford smaller vesicle size,better stability and higher encapsulation efficiency with stronger in vitro anti-tumor activity.

A new mitochondria-targeted platinum complex Mor-platin inhibits HepG2 cell prolif-eration and cell invasion / 中国肿瘤临床

Objective:To investigate the effects of Mor-platin, a novel mitochondrial platinum complex, on proliferation and migration of human hepatoma carcinoma HepG2 cells. Methods:Cell counting kit-8 (CCK-8) assay was used to analyze cell proliferation of Mor-platin and classic anticancer drugs, particularly cisplatin, in HepG2 cells. A laser confocal microscope was used to observe whether Mor-platin can target mitochondria. The morphological changes in cellular mitochondria after treatment with Mor-platin were ob-served on a transmission electron microscope. Cell apoptosis was measured by flow cytometry, and cell invasion was evaluated by three-dimensional tumor spheroid model. Results:Mor-platin can inhibit cell proliferation and is dose dependent. The half inhibitory concentration (IC50) of Mor-platin is lower than that of cisplatin. Laser confocal images showed that Mor-platin can target cell mito-chondria and enrich cell mitochondria. Transmission electron microscopy images showed that cell mitochondrial morphology changed after Mor-platin treatment. Furthermore, cell mitochondrial membrane is incomplete and mitochondrial cristae are reduced. Cell apoptosis caused by Mor-platin is dose dependent. The three-dimensional tumor spheroid model showed that the cell areas of the group subjected to Mor-platin treatment are smaller than those of the control group. Conclusion:Mor-platin can target cell mitochon-dria, change the cell mitochondrial morphology, inhibit cell proliferation, and thus promote cell apoptosis. It also showed better anti-cancer effects than cisplatin. Furthermore, Mor-platin can inhibit three-dimensional tumor spheroid invasion. These results suggest that Mor-platin is a potential antitumor drug.

Inhibitory Effect of Total Alkaloids from Lotus Seed on Human Hepatoma Cells / 中国药师

Objective: To study the inhibitory effect of total alkaloids from lotus seed on human hepatoma HepG2 cells.Methods: The effect of total alkaloids from lotus seed on the growth of HepG2 cells was studied by CCK-8 kit.The apoptosis rate of HepG2 cells was detected by flow cytometry.Results: When the action time was the same, with the increase of drug concentration, the inhibitory rate of total alkaloids from lotus seed on HepG2 cells increased, in a dose-dependent manner.At 72 h, the half inhibitory concentration (IC50) of total alkaloids from lotus seed on HepG2 cells was 1.501 μg·ml-1.At the same concentration, the inhibitory rate of the total alkaloids from lotus seed on HepG2 cells increased with the extension of the action time.At 72 h, the inhibition rate of 10 μg·ml-1 total alkaloids from lotus seed reached 72%.After treated with the total alkaloids from lotus seed at different concentrations, the apoptosis rate of HepG2 cells significantly increased in a dose-dependent manner.Compared with the blank control group, the difference was statistically significant (P <0.05), and the apoptosis rate of HepG2 cells was 85.6% treated with 20 μg·ml-1 total alkaloids from lotus seed.Conclusion: The total alkaloids from lotus seed can induce cell apoptosis and inhibit the proliferation of human hepatoma HepG2 cells.

Effect of ultrasound microbubble mediated miRNA delivery on the ability of migration ,invasion and clon-ing of human hepatoma HepG2 cells / 实用医学杂志

Objective To invstigate the effect of ultrasound microbubble mediated miRNA delivery on mi-gration,invasion and cloning ability of human hepatoma HepG2 cells. Methods The migration,invasion and col-ony forming ability of HepG2 cells were measured after transfection with antisense miRNA-21/221 and miRNA-199a mimic via the optimal ultrasound microbubble transfection method. Results Compared with the control group ,the migration ,invasion and cloning ability of cells were significantly inhibited after transfection with miRNA mimics(P < 0.05,respectively),especially for miR-199a(relative cell migration rate was 31.05%,the number of invasive cells were 38.67 ± 4.51 and the number of clones were 105.67 ± 5.86). Conclusion The pres-ent study may provide new ideas and clues for gene therapy and prognosis of hepatocell ular carcinoma through ana-lyzing the effect of miRNAs on the biological characteristics of human hepatoma HepG2 cells.

Synthesis of 8-bromo-ethoxy Rhein and evaluation of its inhibition effect on hepatitis B virus in human hepatoma cells HepG2.2.15 / 中国药理学通报

Aim To synthesize 8-bromo-ethoxy Rhein and investigate its mechanisms and inhibition effect on hepatitis B surface antigen ( HBsAg ) and e antigen ( HBeAg) in HepG2.2.15 cells.Methods 8-bromo-ethoxy Rhein was synthesized based on the chemical structure of Rhein , and its structure was identified by IR,1 H-NMR and 13 C-NMR spectra.MTT assay was used to test the inhibitory effect of 8-bromo-ethoxy Rhein on HepG2.2.15 cells.After the cells treatment by 8-bromo-ethoxy Rhein , the HBsAg and HBeAg in cell supernatant were detected by ELISA .The expres-sion of hepatitis B virus X gene ( HBx) was detected by Western blot .The cell cycles were examined with flow cytometry.The intracellular free calcium concentration was detected by laser scanning confocal microscopy . Results The structure of 8-bromo-ethoxy Rhein was confirmed by IR,1 H-NMR and 13 C-NMR.MTT results showed that synthetic product and Rhein could inhibit the cell proliferation in HepG2.2.15 cells.After trea-ted with 8-bromo-ethoxy Rhein and Rhein for 72 h,the half inhibitory concentration 50%( IC50 ) was 14.29 mg? L-1 and 11.59 mg? L-1 , respectively .Using non-cytotoxic dose of 8-bromo-ethoxy Rhein , the inhibitory effect on HBsAg and HBeAg was gradually enhanced with increasing 8-bromo-ethoxy Rhein concentration . The inhibitory effect of synthetic product on hepatitis B virus was better than that of Rhein .8-bromo-ethoxy Rhein could down-regulate the expression of HBx , in-tracellular calcium ion concentration and block the hepatitis B virus ( HBV ) replication.Flow cytometry results showed 8-bromo-ethoxy Rhein didn′t affect the cell cycle .Conclusions Compare with Rhein , the synthesis of 8-bromo-ethoxy Rhein shows stronger inhi-bition on hepatitis B virus in HepG2.2.15, and its mechanisms may involve down-regulating the expres-sion of HBx and reducing calcium ion concentration .

Exposure of human hepatoma cells to nitrite and ammonia promotes invasive activity through activation of ROS/ODC pathway / 药学学报

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.

Intervention Effects of(E)-4-[2-(4-Chlorophenoxy)-2-Methylpropanoyloxy]-3-Methoxyphenyl Acrylic Acid on Fat Accumulation in Human Hepatoma HepG2 Cells / 中国药房

OBJECTIVE:To investigate the intervention effects of (E)-4-[2-(4-chlorophenoxy)-2-methylpropanoyloxy]-3-me-thoxyphenyl acrylic acid (AZ) on fat accumulation in human hepatoma HepG2 cells. METHODS:Oleic acid was used to induce fat accumulation in HepG2 cells in logarithmic phase for establishing models of fat accumulation,which were divided into a model group,a positive control group(100 μg/ml simvastatin),and the groups of 15.63,31.25,62.5,125,250,500 and 1 000 μg/ml AZ,and a normal control group was set up. MTT method was used to detect the survival rates of all groups of cells,kit was per-formed to determine the contents of triglyceride (TG) in all groups of cells and calculate the clearance rates,and oil red O stain was conducted to observe the lipid droplet morphology of all groups of cells. RESULTS:Compared to the normal control group, the model group and the groups of 15.63-125 μg/ml AZ demonstrated no obviously different survival rate of cells,and the groups of 250-1 000 μg/ml AZ had lower survival rate of cells. There was statistically significance (P<0.05). The contents of TG in the cells of the model group were higher than those in the cells of the normal control group. The positive control group and the groups of 62.5 and 125 μg/ml AZ had lower contents of TG in the cells compared to the model group,showing a TG clearance rate of (28.58 ± 0.15)%,(14.51 ± 0.09)% and (29.72 ± 0.16)% respectively. There was statistically significance (P<0.01 or P<0.05). There were much more lipid droplets in the cells of the model group than in those of the normal control group. The lipid droplets in AZ groups gradually became less in quantity and smaller with the increasing in drug concentration. CONCLUSIONS:AZ has inter-vention effect on fat accumulation in HepG2 cells.

Effects of proliferation and apoptosis of phloretin on the hepatoma carcinoma cells HepG-2 / 中国生化药物杂志

Objective To investigate proliferative and apoptotic effects of phloretin on hepatoma carcinoma cells, hepatoma carcinoma cells HepG-2 was used as research materials.Methods This research observed morphological alterations using phase contrast microscopy and electron microscopy, cell proliferation were detected by MTT assay, and using flow cytometry detected apoptotic rates, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis.ResuIts Apoptotic cells appeared morphological alterations.Phloretin exerted a inhibitory the proliferation of HepG-2 cell line, and induced its apoptosis in a dosage and duration dependent manner.Cell cycle was arrested at G1 phase, mitochondrial trans-membrane potential dropped, intracellular free Ca2 + increased.ConcIusion Phloretin can induce apoptosis of HepG-2 via arresting cell cycle progression, reducing mitochondrial trans-membrane potential and disturbing intracellular calcium homeostasis.

Mechanism of phloretin induced the apoptosis of hepatoma carcinoma cell SMMC-7721 / 中国生化药物杂志

[Abstract ] Objective To investigate effect of phloretin on apoptotic of hepatoma carcinoma cells SMMC-7721, and explore its mechanisms.Methods Logarithmic phase of hepatoma carcinoma cells SMMC-7721 were cultured separately with 30, 60, 120 mg/L phloretin, morphological alterations of apoptotic were observed by phase contrast microscopy and AO/EB double fluorescence staining method was used to observe were low, medium and high concentration trentment group, respectively.the cells treated by phloretin.Apoptotic rates, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis were detected by flow cytometry.Results Cells appeared typical apoptosis morphological alterations.Phloretin induced SMMC-7721 cell line apoptosis in a dosage and duration dependent manner.Cell cycle was arrested at G1 phase, mitochondrial trans-membrane potential decreased, intracellular free Ca2 +increased.Conclusion Phloretin induce apoptosis of SMMC-7721 by affecting cell cycle progression, reducing mitochondrial trans-membrane potential and changing intracellular calcium homeostasis.