Anti-hepatocellular Carcinoma Activity and Related Mechanism of Apigenin-7-O-Glucoside / 肿瘤防治研究

Objective To investigate the inhibitory effect of apigenin-7-o-glucoside (AGL) on the viability of Huh7 cells and tumor growth in Huh7-xenograft tumor nude mice. Methods CCK-8 was used to detect the proliferation inhibitory effect and the half inhibitory concentration of AGL on Huh7 cells. The mitochondrial membrane potential measurement was used to analyze the early apoptosis of Huh7 cells after AGL treatment. Flow cytometry was used to analyze the effect of AGL on Huh7 cell apoptosis, and Western blot was used to explore the expression level of the proteins associated with apoptosis and inflammation, as well as the possible related mechanism. In Huh7-xenograft tumor nude mice, vernier caliper was used to measure tumor volume to analyze the effect of AGL on tumor growth rate. HE staining was used to observe the pathological state of mouse organs, and the inflammation-related factors in serum were detected with ELISA. Results After Huh7 cells were treated with AGL, the mitochondrial membrane potential reduced, the content of ROS increased and the apoptosis rate was increased to 25.23% by 50 μmol/L AGL treatment; while the expression levels of Bax, Bad, Cleaved Caspase-3 and Cleaved Caspase-9 increased, and the expression levels of Bcl2 and Bcl-xL decreased, the phosphorylation level of NF-κB, IKKα/β and IκBα decreased; the tumor growth rate decreased, the serum IL-6 and TNF-α levels significantly decreased, while the IL-2 and IL-10 levels increased. Conclusion AGL could promote the apoptosis of Huh7 cells and relieve the tumor development in Huh7-xenograft tumor nude mice, which may be related to the NF-κB pathway.

Study on synergistic anti-tumor effect of Shuangdan Capsules combined with 5-FU on hepatocellular carcinoma cells Huh-7 and xenograft mice / 中国中药杂志

This paper discussed the synergistic anti-tumor effect of Shuangdan Capsules combined with 5-fluorouracil(5-FU) on human liver cancer cell line Huh-7 and tumor bearing mice. The effects of Shuangdan Capsules combined with 5-FU on the activity and vascular endothelial growth factor(VEGF) receptor protein expression of Huh-7 cells were investigated, and the effects of drug combination on tube formation of HUVEC cell were also verified. In addition, the mice model of Huh-7 was established to observe the anti-tumor effect of drug combination and the distribution of tumor blood flow in tumor bearing mice by using molecular imaging. HPLC analysis showed that Shuangdan Capsules mainly consisted of danshensusodium, protocatechuic aldehyde, paeoniflorin, rosmarinic acid, alkannic acid, salvianolic acid B, and paeonol. In MTT experiment, the inhibition rate of Shuangdan Capsules(20 mg·L~(-1)) and 5-FU(1 μmol·L~(-1)) on Huh-7 cells was 60%, and the CI value was 0.59, suggesting that these two drugs had synergistic anti-hepatoma cells effect. The expression of VEGF receptor in Huh-7 cells was inhibited by the combination of these two drugs. In addition, the process of HUVEC was slow, and the number, length and area of the lumen branches decreased significantly. In vivo, Shuangdan Capsules combined with 5-FU inhibited the growth and prolongation of survival of Huh-7 cells in subcutaneous transplanted tumor nude mice; serum expression of CD31 and VEGF in nude mice were decreased, while caspase-3 was increased. Meanwhile, the drug combination significantly inhibited the expressions of MMP2 and VEGF in tumor tissues. Ultrasound showed that Shuangdan Capsules combined with 5-FU also inhibited tumor angiogenesis and reduced blood flow of tumor tissue. The results showed that Shuangdan Capsules combined with 5-FU may inhibit tumor angiogenesis by inhibiting VEGF and MMP2 expressions, thereby blocking tumor growth.

Study on Improvement Effect of Methylated Urolithin A on Oleic Acid-induced Lipid Accumulation in Huh- 7 Cells and Its Me- chanism / 中国药房

OBJECTIVE： To study the improvement effect and mechanism of methylated urolithin A on oleic acid-induced lipid accumulation in human liver cancer Huh-7 cells. METHODS： Oleic acid was adopted to induce lipid accumulation model cells. Huh-7 cells were divided into control group （culture medium）， model group （1 mmol/L oleic acid）， low-dose group （1 mmol/L oleic acid+10 μmol/L methylated urolithin A） and high-dose group （1 mmol/L oleic acid+20 μmol/L methylated urolithin A）. Oil red O staining was used to observe lipid accumulation in cells. Triglyceride（TG） enzyme assay was applied to determine the TG content in cells. PCR was employed to detect the mRNA expression of FASN， SREBP-1， PPAR-α and PPAR-γ in cells. Western blotting was used to determine the protein expression of FASN in cells. RESULTS： After induced by oleic acid， a large amount of lipid droplet accumulated around the cells； the intracellular lipid and TG content， mRNA expression levels of FASN， SREBP-1 and PPAR-γ， protein expression levels of FASN were increased significantly， while mRNA expression level of PPAR-α was decreased significantly （P＜0.01）. After intervened with methylated urolithin A， lipid droplet around the cells decreased significantly； the contents of lipid and TG in cells were decreased significantly， while the mRNA expression levels of FASN， SREBP-1 and PPARγ and protein expression level of FASN were decreased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： Methylated urolithin A can improve oleic acid-induced lipid accumulation in Huh-7 cells， the mechanism of which may be associated with inhibiting fat synthesis， promoting lipid metabolism and down-regulating the expression of metabolism-related factors as FASN， SREBP-1 and PPAR-γ.

Effect and mechanism of dauricine on proliferation and apoptosis of hepatoma Huh7 cells / 中草药

Objective To study the effect of dauricine on the proliferation and apoptosis of hepatoma Huh7 cells, and explore its anti-tumor mechanism and its relationship with Hedgehog signaling pathway. Methods The effects of different concentrations of dauricine (2, 4, 8 μg/mL) on the proliferation of Huh7 cells were detected by MTT assay. Apoptosis of Huh7 cells was analyzed by flow cytometry. Real-time PCR and Western blotting were used to detect the levels of Hedgehog signaling pathway-related genes and proteins. Results With the increase of the concentration of dauricine and the duration of action, the inhibition rate of Huh7 cell proliferation was increased. Among them, 8 μg/mL dauricine had the highest inhibition rate (48.8%) at 48 h. Dauricine induced the apoptosis in Huh7 cells. With the increase of the concentration of dauricine, the apoptotic rate of cells was increased significantly (P < 0.05, 0.01). The mRNA and protein expression levels of PTCH1, GLi1, SMO and SHH genes in Hedgehog signaling pathway were significantly decreased, while the level of cleaved Caspase-3 protein was significantly increased, accompany with the decreased expression of Bcl-2 in dauricine concentration-dependent pattern (P < 0.05, 0.01) in dauricine group compared with the control group. Conclusion Dauricine could significantly inhibit the proliferation and promote apoptosis of Huh7 cells, which may play a role by blocking Hedgehog signaling pathway.

Effect of Compound Phyllanthus urinaria Ⅱ(CPU Ⅱ) on Proliferation, Apoptosis and Autophagy of Hepatocellular Carcinoma Huh7 Cells / 中国实验方剂学杂志

Objective:To observe the effect and investigate the mechanism of compound Phyllanthus urinaria Ⅱ(CPU Ⅱ)on proliferation, apoptosis and autophagy of human hepatoma cell line Huh7. Method:Huh7 cells were cultured in vitro and divided into blank control group, high-dose CPU Ⅱ group (40 g·L-1),low-dose CPU Ⅱ group (20 g·L-1), and 5-FU group (0.04 g·L-1). Methye thiazolye telrazlium(MTT) assay was used to detect the inhibitory effect of CPU Ⅱ on proliferation of human hepatoma Huh7 cells. The apoptosis rate was observed by Annexin V-FITC/PI flow cytometry; the changes of autophagosomes in each group were observed by monodansylcadaverin (MDC) staining; Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the mRNA expression of phosphatidylinositol 3-kinase (PI3K), Serine-threonine protein kinase 2(Akt2), B-cell lympoma-2(Bcl-2) and Microtubule-associated protein 1 light chian 3(LC3Ⅱ) and Western blot was used to detect the protein expression of Akt2 and LC3Ⅱ. Result:CPU Ⅱ(40, 20 g·L-1) significantly inhibited the hepatoma cell line Huh7 and induced apoptosis, with an apoptosis rate of 51.72% and 19.74% respectively, significantly higher than that of control group (PPPPConclusion:CPUⅡ had obvious inhibitory effect on the proliferation of hepatoma cell line Huh7, and the mechanism may be related to inhibiting the activation of PI3K/Akt signaling pathway to induce apoptosis and autophagy.

Effect of cinobufagin combined with Sorafenib on proliferation and apoptosis of hepatocellular carcinoma Huh7 cells via AURKA/Ras/Raf/ERK signaling pathway / 中草药

Objective To investigate the effect and mechanism of cinobufagin combined with Sorafenib on the proliferation and apoptosis of hepatocellular carcinoma Huh7 cells. Methods The proliferation of Huh7 cells was measured using MTT assay; The apoptosis morphological changes of Huh7 cells were detected using Hoechst33342/PI fluorescence staining; The cells cycle was detected by flow cytometry; The expression of Ki67 protein was detected by immunocytochemistry; The expressions of Bax, Bcl-2, Caspase-8, AURKA, Ras, Raf, ERK, and p-ERK proteins were measured using Western blotting. Results Cinobufagin, Sorafenib, and combination therapy inhibited the proliferation of Huh7 cells, and the inhibitory effect of the combination group was more obvious with synergistic effect. Fluorescence staining showed morphological changes of apoptosis. Sorafenib induced the cell cycle S phase arrest, cinobufagin and combination therapy induced the cell cycle G2/M phase arrest, combination group had more obvious cell cycle arrest in G2/M phase than single drug groups. Both cinobufagin and Sorafenib attenuated the expression of Ki67, and the effect of combination group was more significant. Cinobufagin, Sorafenib, and combination therapy up-regulated the expression of Bax and Caspase-8 proteins; down-regulated the expression of Bcl-2 protein; up-regulated the ratio of Bax/Bcl-2; had no obvious effect on the expression of ERK protein; significantly down-regulated the expression of AURKA, Ras, Raf, and p-ERK proteins; And the effect of combination group was more significant (P < 0.05). Conclusion Cinobufagin combined with Sorafenib could inhibit the proliferation and induce the apoptosis of hepatocellular carcinoma Huh7 cells through AURKA/Ras/Raf/ERK signaling pathway.

Cissampelos sympodialis has anti-viral effect inhibiting dengue non-structural viral protein-1 and pro-inflammatory mediators

ABSTRACT Dengue is the most important viral infection transmitted among humans by arthropod-borne. There are currently no vaccines or specific therapeutical treatment. Therefore, immunomodulatory compounds from plants have been widely examined for their antiviral effects. Cissampelos sympodialis Eichler, Menispermaceae, has scientifically proven to present immunomodulatory activities. Here we assessed the antiviral activity of leaf hydroalcoholic extract, warifteine or methylwarifteine from C. sympodialis in an in vitro dengue virus infection model. The results demonstrated that leaf hydroalcoholic extract or warifteine/methylwarifteine treatment did not reduce dengue virus-Ag+ hepatocyte (Huh-7 cell) rates in present experimental conditions. However, we assessed the potential antiviral effect of leaf hydroalcoholic extract or warifteine/methylwarifteine on dengue virus-infection by the production of inflammatory molecules, TNF-α, MIF, IL-8 and PGE2. Dengue virus infection enhanced TNF-α, MIF, IL-8 and PGE2 production in infected Huh-7 cells and leaf hydroalcoholic extract but not warifteine/methylwarifteine treatments, significantly reduced these molecules in infected cells. In dengue virus-infected Huh-7 cells, non-structural protein-1 is produced and leaf hydroalcoholic extract significantly inhibited it independently of alkaloids. Our findings imply that leaf hydroalcoholic extract may attenuate dengue virus infection in Huh-7 cells by inhibiting the enhanced of pro-inflammatory mediators and non-structural protein-1 production induce by dengue virus independently of warifteine/methywarifteine its major compound.

Effect of GPR49 gene on proliferation and invasive ability of hepatoma cells / 器官移植

Objective To discuss the effect of G-protein-coupled receptor 49 (GPR49)gene on proliferation and invasive ability of hepatoma cell line Huh7 and its molecular biological mechanism.Methods According to the different transfected small interfering RNA(si-RNA),Huh7 cells were divided into the GPR49-siRNA(si-GPR49)group and the NC-siRNA (si-NC)group.Untransfected Huh7 cells were set as the control group. Messenger RNA (mRNA )and protein expression of GPR49, cyclin D1 and matrix metalloproteinase 9 (MMP9)in the cells of the three groups were respectively detected by reverse transcription-polymerase chain reaction (RT-PCR)and Western blot method.The proliferation and invasive ability of the cells of each group were respectively detected by MTT method and Transwell method.Results The relative expression of GPR49 mRNA of the si-GPR49 group was (23.8 ±3.1)% of the control group (P <0.05). Compared with the control group,the protein expression of GPR49,cyclin D1 and MMP9 of the si-GPR49 group decreased significantly (all in P <0.05).The proliferation experiment results by MTT indicated that the optical density(OD)of the cells of the si-GPR49 group at 72 h was (0.53 ±0.12),which was significantly lower than that of the control group (1.35 ±0.28).The difference had statistical significance (P <0.05). The average invaded cell counts of the si-GPR49 group were (13.6 ±2.5),which was significantly lower than (65.3 ±6.1 )of the control group.The difference had statistical significance (P <0.05 ).Conclusions GPR49-siRNA may inhibit the gene expression of GPR49 in Huh7 cells.Its mechanism may be that the proliferation of Huh7 cells is inhibited by reducing the level of cyclin D1;the migration and invasive ability of Huh7 cells is inhibited by affecting the expression level of MMP9.

Chk1 gene scijencing potentiates human hepatoma Huh7 cells to curcumin-induced apoptosis / 中国癌症杂志

Background and purpose: Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radio- or chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chkl/2as a therapeutic target to potentiate human hepatoma to curcumin. Methods: Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Westem blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Westem blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results: Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P<0.01). Inhibition of Chk1, but not Chk2, increased apoptotic rate from (21.3±1.8)% to (29.5±2.6)% (P<0.05). Neither Chk1 nor Cbk2 siRNA had any impact on cell cycle distribution in Huh7 cells induced by curcumin. Conclusion: Chk1 siRNA sensitized Huh7 cells to curcumin-induced apoptosis, suggesting that Chk1 is a potential therapeutic target to sensitize human hepatoma to curcumin.

Chk1 gene scilencing potentiates human hepatoma Huh7 cells to curcumin-induced apoptosis / 中国癌症杂志

Background and purpose:Checkpoint kinase 1 and 2 have been proposed to be potential therapeutic targets to sensitize cancers to radioor chemo-therapeutics. However, little is known about whether Chk1/2 is also a suitable target for sensitizing cancers to curcumin. In the present study, we investigated effects of Chk1/2 siRNA on curcumin-induced apopotosis in hepatoma cell line Huh7 and evaluated the effectiveness of Chk1/2 as a therapeutic target to potentiate human hepatoma to curcumin. Methods:Effect of curcumin on the cell cycle checkpoint-associated proteins was detected by Western blot. The knockdown efficacy of Chk1/2 siRNA was measured by RT-PCR and Western blot. Effect of Chk1/2 siRNA on curcumin-induced apoptosis in Huh7 cells was evaluated by DAPI staining. Effect of Chk1/2 siRNA on cell cycle distribution in curcumin-treated Huh7 cells was analyzed by flow cytometry. Results:Curcumin significantly inhibited phosphorylation of cell cycle checkpoint-associtaed proteins Chk1(S317), Cdc25C(S216) and Cdk1(Y15). Chk1 siRNA decreased Chk1 mRNA and protein by 95% and 92% and Chk2 siRNA decreased Chk2 mRNA and protein by 60% and 55% respectively as compared with negative control siRNA (P