Human Hepatoma SMMC-7721 Cells Research Articles

Efficient separation of five flavonoids from Oxytropis falcata bunge by high-speed counter-current chromatography and their anticancer activity

Oxytropis falcata Bunge, known as the “king of herbs” in Tibetan medicine, is used for treatment of hyperpyrexia, pain, wounds, inflammation, and anthrax. However, it is difficult to efficiently isolate compounds with high purity from O. falcata because of the complexity of traditional Tibetan medicines. In this study, the 80% ethanol elution fraction from extract by AB-8 macroporous resin column chromatography was demonstrated to have anticancer activity on human hepatoma SMMC-7721 cells in vitro. Then, a high-speed counter-current chromatography (HSCCC) method was successfully established for separation of compounds by using hexane–ethyl acetate–methanol–water (10:4:10:10, v/v/v/v) as the solvent system. Five flavonoids (7-hydroxyflavonone [1], 5,7-dihydroxy-4′-methoxy flavonol [2], 5,7-dihydroxyflavanone [3], 2′,4′-dihydroxychalcone [4], and 2′,4′-dihydroxydihydrochalcone [5]) were obtained in one-step separation with purities of 97.7%, 98.1%, 98.3%, 99.0%, and 98.3%, respectively. Finally, anticancer activities against the growth of SMMC-7721 cells of 5 flavonoids were confirmed. The IC50 values of the separated compounds were 213.45 μg/mL, 197.74 μg/mL, 375.16 μg/mL, 17.44 μg/mL, and 136.83 μg/mL in 24 h, respectively. The present study provided a basis for further development and utilization of this medicinal herb as a source of a new potential anticancer agent.

The Novel Nature Microtubule Inhibitor Ivalin Induces G2/M Arrest and Apoptosis in Human Hepatocellular Carcinoma SMMC-7721 Cells In Vitro.

Background and Objectives: Microtubules are an attractive target for cancer chemotherapy. Previously, we reported that Ivalin exhibited excellent anti-migration and anti-invasion activities in human breast cancer cells. Here, we examined the microtubule inhibition effect of Ivalin in human hepatocellular carcinoma SMMC-7721 cells. Materials and Methods: We used the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the cell proliferation effect of Ivalin and flow cytometry analysis to detect the apoptotic and cell cycle arrest effects of Ivalin. Immunofluorescence staining was used to measure the effect of Ivalin on the cytoskeleton network, and Western blotting was used to detect the expression levels of Bax, Bcl-2, Cdc2, phosphor-Cdc2, Cdc25A, Cyclin B1, and tubulin. Results: Ivalin induced cell cycle G2/M arrest and subsequent triggered apoptosis in human hepatocellular carcinoma SMMC-7721 cells. Furthermore, microtubules were shown to be involved in Ivalin-meditated apoptosis. In this connection, Ivalin treatment suppressed cellular microtubule network formation by regulating microtubule depolymerization. Moreover, Western blotting revealed Cdc25A and Cyclin B1 were upregulated in Ivalin-meditated cell cycle arrest. Subsequently, the induction of Bax (a proapoptotic protein) and reduction of Bcl-2 (an anti-apoptotic protein) expression were observed in Ivalin-treated SMMC-7721 cells. Conclusion: Ivalin induced microtubule depolymerization, then blocked cells in mitotic phase, and eventually resulted in apoptosis in SMMC-7721 cells. Collectively, these data indicate that Ivalin, acting as a novel inhibitor of microtubules, could be considered as a promising lead in anticancer drug development.

Intestinal probiotics E. coli Nissle 1917 as a targeted vehicle for delivery of p53 and Tum-5 to solid tumors for cancer therapy

Traditional cancer therapies, such as surgery treatment, radiotherapy, and chemotherapy, often fail to completely eliminate tumor cells in an anaerobic microenvironment of tumor regions.In contrast to these traditional cancer therapies, the use of targeted delivery vectors to deliver anticancer genes or antitumor drugs to hypoxic areas in tumors is the most clinically promising cancer treatment with rapid development in recent years. In this study, E.coli Nissle 1917 (EcN), an intestinal probiotic, was utilized as a targeted transport vector to deliver p53 and Tum-5 protein to tumor hypoxic regions. The tumor-targeting characteristics of EcN were investigated using luciferase LuxCDABE operon, and the results demonstrated that EcN could specifically accumulate in the solid tumor areas of SMMC-7721 tumor-bearing BALB/c nude mice. The Tum 5-p53 bifunctional proteins were initially constructed and then delivered to solid tumor regions by using the targeted transporter EcN for cancer therapy. The antitumor effect and safety of three engineered bacteria, namely, EcN (Tum-5), EcN (p53), and EcN (Tum 5-p53), were also examined. The calculated tumor volume and tumor weight indicated that these three engineered bacteria could inhibit the growth of human hepatoma SMMC-7721 cells, and the antitumor effect of EcN (Tum 5-p53) expressing the Tum 5-p53 fusion protein was significantly better than those of EcN (Tum-5) and EcN (p53) alone. Immunofluorescence demonstrated that the expression of Ki-67, a nuclear proliferation-related protein, was inhibited in the tumor areas of the groups treated with the engineered bacteria, whereas the expression of caspase-3 was upregulated. The expression trends of Ki-67 and caspase-3 were consistent with the different antitumor efficacies of these three engineered bacteria. EcN did not elicit obvious side effects on mice. This research not only provids a foundation for tumor-targeted therapy but also contributes greatly to the development of antitumor agents and anticancer proteins.

Uncovering the anti-metastasis effects and mechanisms of capsaicin against hepatocellular carcinoma cells by metabolomics

Abstract Capsaicin, a kind of alkaloid from pepper cortex, has extensive anti-tumor activities. However, the anti-metastasis effects and mechanisms of capsaicin against hepatocellular carcinoma (HCC) have not been deeply studied so far. In the present work, the anti-metastasis effects of capsaicin were investigated in human hepatoma SMMC-7721 and HepG2 cells, and its mechanisms were explored using metabolomics-based approach. The results showed that capsaicin remarkably suppressed cell proliferation, migration, and invasion, and hampered cell-matrix adhesion in SMMC-7721 and HepG2 cells. Metabolomics approach revealed that 18 differential metabolites, and 5 metabolic pathways including glutathione metabolism, pyruvate metabolism, phenylalanine metabolism, glycine, serine and threonine metabolism, glycerophospholipid metabolism were implicated in the anti-HCC effects of capsaicin. A multi-level network further uncovered the possible molecular mechanisms of capsaicin in regulating these metabolites and metabolic pathways. This study revealed that metabolomics integrating with network-based approach can facilitate understanding of the anti-metastasis mechanisms of capsaicin against HCC.

Design, synthesis, and biological activity evaluation of (-)-6-O-desmethylantofine analogues as potent anti-cancer agents.

Phenanthroindolizidine alkaloids that possess profound anti-proliferative activity and unique mode of action have recently attracted much attention as potential anti-cancer drug candidates. To intensively study the structure-activity-relationship, we designed, synthesized, and evaluated a series of derivatives of 6-desmethylantofine at C-6 position. Most of the derivatives exhibited potent anti-proliferative activity in BEL-7402 and HL60cells. Compound R-12, the cyanomethyl ether of 6-desmethylantofine, exhibited significant anti-cancer activity and inhibited the proliferation of a panel of 30 cancer cell lines including 2 multi-drug-resistant cell lines with an average IC50 value of 18.7 nM, which suggests that R-12 is a promising new anti-cancer agent. Our studies suggest that R-12 displayed potent inhibitory effect on cell growth and colony formation, which is associated with delaying S phase progression by inhibiting DNA synthesis in human hepatoma cancer BEL-7402, SMMC-7721 and ZIP-177 cells.

Synthesis, Crystal Structure and Antitumor Activity of 2-(5,6-Dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-6-ethoxyphenol

A new 2-(2’-aminophenyl)benzimidazole derivative, 2-(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-6-ethoxyphenol (1), was synthesized by the reaction of 2-(2’-aminophenyl)benzimidazole with 3-ethoxybenzaldehyde. Its structure has been determined by elemental analysis and X-ray single crystal diffraction analysis. Compound 2 crystallizes in triclinic, space group P-1 with a = 8.7010(17) Å, b = 11.022(2) Å, c = 11.470(2) Å, α = 107.11(3)°, β = 108.96(3)°, γ = 104.61(3)°, V = 917.7(3) Å3, Z = 2, Dc = 1.293 g·cm−3, μ = 0.085 mm−1, F(000) = 376, and final R1 = 0.0439, ｗR2 = 0.1214. The antitumor activities of compound 1 against human hepatoma SMMC-7721 cells and human colon carcinoma WiDr cells have been investigated.

Synthesis and crystal structure of bicopper(II) complexes: The influence of bridging ligands on DNA/BSA binding behaviors and in vitro antitumor activity

Two new μ-oxamide or μ-oxamate bridged bicopper(II) complexes, [Cu2(mapox)(bpy)2](ClO4)2 (1) and Cu2(mapoa)(CH3OH)(bpy)2(pic)2·0.5H2O (2) (H2mapox = N,N′-bis[3-(methylamino)propyl]-oxamide, H2mapoa = N-[3-(methylamino)propyl]oxamate, bpy = 2,2′-bipyridine, pic = picrate anions), have been synthesized and characterized by elemental analyses, molar conductance measurement, spectroscopic studies and single-crystal X-ray diffraction. In complex 1, both the copper(II) atoms acquire a square-pyramidal coordination geometry. The distance between Cu1 and Cu2 atoms is 5.2541(8) Å. The structure of complex 2 contains two bicopper(II) cations [Cu2(mapoa)(CH3OH)(bpy)2]2+, in which the Cu atoms acquire distorted square-pyramid coordination geometry (Cu1, Cu2, Cu4) or distorted trigonal bipyramid coordination geometry (Cu3). The Cu⋯Cu separations are 5.2316(8) Å (Cu1, Cu2) and 5.2187(7) Å (Cu3, Cu4), respectively. The interaction between complexes 1 and 2 and herring Sperm DNA (HS-DNA) were studied by using UV absorption and fluorescence spectra, electrochemical techniques and viscometry. The results indicated that both of the two complexes could bind to HS-DNA by intercalation with the order 1 > 2. Protein binding properties investigations reveal that the two complexes could bind to bovine serum albumin (BSA) by the static quenching mechanism. The in vitro cytotoxicities experiments reveal that complex 1 exhibit better growth-inhibitory effects than complex 2 on human hepatocellular carcinoma cell SMMC-7721 and human lung adenocarcinoma cell A549.

Role of miR-16-5p in the proliferation and metastasis of hepatocellular carcinoma.

The aim of this study was to investigate the role of miR-16-5p in hepatocellular carcinoma (HCC), and to explore the possible underlying mechanism. 100 pairs of cancerous and para-cancerous tissues surgically removed in our hospital were collected. Real Time quantitative-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression level of miR-16-5p in tissues. Bioinformatics and Dual-Luciferase reporter gene assay were used to screen and verify the potential target genes of miR-16-5p, respectively. Human HCC SMMC-7721 cells were used for functional experiments. Cell proliferation was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Cell invasion and migration were evaluated by transwell and scratch wound-healing assay, respectively. The protein expression levels of epithelial-mesenchymal transition (EMT) associated markers were measured by Western blot (WB) assay. QRT-PCR showed that miR-16-5p expression in HCC tissues was significantly lower than that of adjacent normal liver tissues. At the cellular level, miR-16-5p was lowly expressed in HCC cells (SMMC-7721). Bioinformatics websites (including Targetscan, PicTar, miRanda) predicted that insulin-like growth factor 1 receptor (IGF1R) was a potential target gene of miR-16-5p. Meanwhile, IGF1R was selected for further investigation due to its metastatic function. The results showed that no significant difference was found in the mRNA expression level of IGF1R in HCC tissues. However, the protein level of IGF1R was significantly up-regulated, which was negatively correlated with miR-16-5p. Combined with Dual-Luciferase reporter gene assay, it was confirmed that miR-16-5p could regulate the expression of IGF1R in a targeted manner. Furthermore, down-regulation of IGF1R significantly reduced the inhibitory effect of miR-16-5p on the proliferation and metastasis of SMMC-7721 cells. We showed that miR-16-5p suppressed invasion and migration of HCC cells, mechanically by directly targeting and inhibiting IGF1R protein expression. The newly identified miR-16-5p/IGF1R axis might provide new insights into the pathogenesis of HCC and novel potential therapeutic targets for the treatment of HCC.

Gemfibrozil reduces lipid accumulation in SMMC-7721 cells via the involvement of PPARα and SREBP1.

Gemfibrozil (GEM) is a member of the fibrate class of lipid-lowering pharmaceuticals and has been widely used in the therapy of different forms of hyperlipidemia and hypercholesterolemia. Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease and is becoming an important public health concern worldwide. However, there is little knowledge about the effects of GEM on NAFLD. In the present study, oleate-treated human hepatoma SMMC-7721 cells were utilized to investigate the role of GEM in regulating hepatic lipid metabolism. The present results demonstrated that GEM attenuated excessive intracellular triglyceride content in the steatosis model. Upregulation of peroxisome proliferator-activated receptor α (PPARα) protein and sterol regulatory element-binding protein 1 (SREBP1) was detected following treatment with GEM. Additionally, reverse transcription-polymerase chain reaction analysis demonstrated that GEM increased the downstream genes related to PPARα and SREBP1, including carnitine palmitoyltransferase 2, acyl-coA oxidase 1, hydroxyacyl-CoA dehydrogenase, LIPIN1 and diacylglycerol O-acyltransferase 1. These findings demonstrated that GEM alleviated hepatic steatosis via the involvement of the PPARα and SREBP1 signaling pathways, which enhances lipid oxidation and interferes with lipid synthesis and secretion. Taken together, the data provide direct evidence that GEM may lower lipid accumulation in hepatocellular steatosis cells in vitro and that it may have a potential therapeutic use for NAFLD.

Hispidulin induces ER stress-mediated apoptosis in human hepatocellular carcinoma cells in vitro and in vivo by activating AMPK signaling pathway.

Hispidulin (4',5,7-trihydroxy-6-methoxyflavone) is a phenolic flavonoid isolated from the medicinal plant S. involucrata, which exhibits anti-neoplastic activity against several types of cancer. However, the mechanism underlying its anti-cancer activity against hepatocellular carcinoma (HCC) has not been fully elucidated. In this study, we investigated whether and how hispidulin-induced apoptosis of human HCC cells in vitro and in vivo. We showed that hispidulin (10, 20 μmol/L) dose-dependently inhibited cell growth and promoted apoptosis through mitochondrial apoptosis pathway in human HCC SMMC7721 cells and Huh7 cells. More importantly, we revealed that its pro-apoptotic effects depended on endoplasmic reticulum stress (ERS) and unfolded protein response (UPR), as pretreatment with salubrinal, a selective ERS inhibitor, or shRNA targeting a UPR protein CHOP effectively abrogated hispidulin-induced cell apoptosis. Furthermore, we showed that hispidulin-induced apoptosis was mediated by activation of AMPK/mTOR signaling pathway as pretreatment with Compound C, an AMPK inhibitor, or AMPK-targeting siRNA reversed the pro-apoptotic effect of hispidulin. In HCC xenograft nude mice, administration of hispidulin (25, 50 mg/kg every day, ip, for 27 days) dose-dependently suppressed the tumor growth, accompanied by inducing ERS and apoptosis in tumor tissue. Taken together, our results demonstrate that hispidulin induces ERS-mediated apoptosis in HCC cells via activating the AMPK/mTOR pathway. This study provides new insights into the anti-tumor activity of hispidulin in HCC.

Expression of Allograft Inflammatory Factor-1 (AIF-1) in Hepatocellular Carcinoma.

BackgroundAllograft inflammatory factor-1 (AIF-1) is a cytoplasmic protein cloned from activated macrophages in human and rat allografts. AIF-1 has been identified as a modulator of inflammatory response, and recently published studies have shown its increased expression in carcinogenesis. However, there are still limited data on the potential functional role of AIF-1 in hepatocellular carcinoma (HCC).Material/MethodsWe evaluated the expression of AIF-1 in 104 cases of paired HCC and adjacent non-cancerous liver tissues using immunohistochemistry, Western blotting, and qPCR analysis, and sought to determine whether its expression was correlated with clinicopathological features. In vitro assays, including cell proliferation and migration assays, were used to study the effects of AIF-1 knockdown in L02 human hepatocyte, and Huh7 and SMMC7721 liver cancer cell lines.ResultsExpression of AIF-1 was increased in HCC compared to adjacent normal liver tissues and was positively correlated with median tumor size (p=0.046), number of tumor deposits (p=0.009), the Barcelona Clinic Liver Cancer (BCLC) stage (p=0.004), and portal vein tumor thrombus (PVTT) (p<0.001). Huh7 and SMMC7721 human HCC cells demonstrated upregulated AIF-1 expression compared to normal hepatocytes. Small interfering RNA (siRNA)-mediated silencing of AIF-1 expression resulted in a reduction in cell proliferation and migration in human HCC cells.ConclusionsThese findings suggest AIF-1 may have roles as a diagnostic or prognostic biomarker and a promising therapeutic target in HCC.

VTIQ evaluates antitumor effects of NET‑1 siRNA by UTMD in HCC xenograft models

The present study used a virtual touch tissue imaging and quantification (VTIQ) method to investigate the change in elasticity in xenograft tumor tissue models following silencing of the neuroepithelial-transforming protein 1 (NET-1) gene by ultrasound-targeted microbubble destruction (UTMD). A total of 24 xenograft models were established by subcutaneous injection of human hepatocellular carcinoma SMMC-7721 cells in BALB/c female nude mice. Then, NET-1 small interfering RNA (siRNA)-conjugated nanobubbles and a glypican-3 antibody were synthesized. The mean and maximum shear wave speed (SWSmean and SWSmax) in the tumor tissue were measured prior to, during, and following therapy using VTIQ. The growth of the tumor size and survival time were recorded. The levels of NET-1 protein were evaluated by immunohistochemical staining. In addition, tumor, liver and kidney tissues of the nude mice were collected to confirm whether gene transfection treatment was toxic in vivo. In the UTMD delivery gene group, SWSmean was correlated with the maximum diameter of the tumor (r=0.9806, P=0.0194). The immunohistochemical staining data indicated that the level of NET-1 protein in the treated groups was significantly decreased compared with those in the control groups. Additionally, no structural damage was observed in the nude mice liver and kidney tissues following treatment. Therefore, VTIQ measurement identified potential changes in the elastic properties of the tumors, which in turn may be associated with the stages of tumor development. The delivery method, UTMD, improves the antitumor effects of NET-1 siRNA and supports gene transfection as a promising therapeutic strategy.

The clinical significance of the expression of breast cancer resistance protein gene in hepatocellular carcinoma tissue and the effect of inhibiting its expression on the proliferation and apoptosis of hepatocellular carcinoma cells

Objective To investigate the relationship between the expression of breast cancer resistance protein (BCRP) and the clinicopathological features in hepatocellular carcinoma (HCC) and the influence of the BCRP expression on the proliferation and apoptosis of HCC cells. Methods To detect the expression of BCRP in hepatocellular carcinoma tissues by reverse transcriptase-polymerase chain reaction (RT-PCR), and analyze the relationship with clinicopathological parameters; BCRP-small interfering RNA (siRNA) was transfected into human hepatoma SMMC-7721 cells by Lipofectamine™ 2000 liposome mediated method; cell proliferation, apoptosis and BCRP, proliferation cell nuclear antigen (Ki-67), B cell lymphoma/leukemia-2 associated X protein (bax), Notch1 and Hes1 protein expression were detected by methyl thiazol tetrazolium (MTT) method, flow cytometry and Western blotting, respectively. Results The expression of BCRP in HCC was not related to the sex, age and tumor size of HCC patients, and it was related to pathological grade, clinical stage and metastasis of tumor; expression of BCRP in SMMC-7721 cells after BCRP-siRNA was transfected cells was inhibited in the level of transcription and translation; cell proliferation (0.287±0.022) and expression of Ki-67 (0.142±0.018), Notch1 (0.162±0.021) and Hes1 (0.111±0.015) protein in BCRP-siRNA group were significantly lower than Control group (0.411±0.026, 0.258±0.026, 0.462±0.032, 0.241±0.023; Pproliferation=0.001, PKi-67=0.001, PNotch1=0.000, PHes1=0.001), the apoptosis rate (14.63±0.88)% and the expression of bax protein (0.324±0.034) was significantly higher than Control group ([1.32±0.15)%, 0.106±0.012; Papoptosis=0.000, Pbax=0.000]. Conclusion The expression of BCRP is related to pathological grade, clinical stage and metastasis of HCC, inhibition of its expression can reduce the proliferation of hepatocellular carcinoma cells and induce cell apoptosis, the mechanism is related to the regulation of Ki-67, bax expression and Notch1 signaling pathway. Key words: Breast cancer resistance protein gene; Clinical significance; Liver cancer; Apoptosis; Signal pathway

The first phytochemical investigation of Rhododendron websterianum: triterpenoids and their cytotoxic activity

The first phytochemical investigation of the fruits of Rhododendron websterianum resulted in the isolation of two new triterpenoids, 11-dehydroxytrisnormogrol acid (1), 11-dehydroxytrisnormogrol (2), and 21 known analogues. Their structures were determined by extensive analysis of spectroscopic and mass data, as well as by comparison with those in literature. All isolates were assayed their cytotoxic activities on human hepatoma SMMC-7721 cells and human lung adenocarcinoma A549 cells. Three known compounds, dammarenediol II, lup-20(29)-ene-2α,3β-diol, and 19,24-dihydroxyurs-12-en-3-one-28-oic acid exhibited cytotoxic activities against these two human cancer cell lines with the IC50 values in a range of 27.4–42.8 μM.

Preparation, Characterization, Toxicity and Pharmacodynamics of the Inclusion Complex of Brucea javanica Oil with β-cyclodextrin Polymers

The novel water-soluble inclusion complex of Brucea javanica oil (BJO) by β-cyclodextrin polymers (CDP) was prepared by saturated aqueous method and characterized by SEM, FT-IR and 1H NMR. Compared with BJO, the aqueous solubility of BJO-CDP (77.76%) greatly enhanced due to the water-soluble CDP host. In the acute toxicity test, the value of LD50 of BJO-CDP was 11.94 g/kg, suggesting the lower toxicity of BJO-CDP. Moreover, the pharmacodynamics of BJO-CDP was investigated by evaluating its inhibition effects on human hepatoma SMMC-7721 cells and mice transplantable colon cancer CT- 26 cells. It has been revealed that BJO-CDP significantly decreased the toxicity of BJO and enhanced its anti-tumor activity. In conclusion, BJO-CDP could be a new and improved clinical formulation of BJO with higher water solubility, lower toxicity and enhanced anti-tumor activity.

Determination of Eupatilin in Folium artemisiae Argyi and Its Inhibitory Effect on Hepatoma Cells.

Aim:The aim of this study is to establish a method for determination of eupatilin in Folium artemisiae Argyi and observe the inhibitory effect of Folium artemisiae Argyi extract on human hepatoma SMMC-7721 cells.Methods:High-performance liquid chromatograph system with 2910 pump, 2930 UV detector, and N2000 workstation was used for determination of eupatilin in Folium artemisiae Argyi. Human hepatoma SMMC-7721 cells were cultured and cell proliferation was measured using the MTT assay. The expression protein levels of p53, Topo II, and bcl-2 were detected using Western blotting.Results:Eupatilin exhibited a linearity range of 0.5–3.0 μg/mL and a recovery of 100.72%, relevant standard derivation = 2.28%. Folium artemisiae Argyi extract had marked cytostatic and cytotoxic effects on SMMC-7721 cells, inhibited the SMMC-7721 colony formation in a dose-dependent manner. Folium artemisiae Argyi extract already possessed delayed effect after treating SMMC-7721 cells for 8 h, which became obvious at 12 h from treatment. After drug withdrawal, cells still tended to apoptosis. Folium artemisiae Argyi extract could inhibit p53, Topo II, and bcl-2 expressions in tumor cells. The present method for determination of eupatilin is simple, fast, accurate, sensitive, and reproducible.Conclusion:Hepatoma SMMC-7721 cells are quite sensitive to Folium artemisiae Argyi extract, which may be associated with its suppression of p53, Topo II, and bcl-2 expressions.SUMMARY The study aimed to establish a method for determination of eupatilin in Folium artemisiae Argyi and observe the inhibitory effect of Folium artemisiae Argyi extract on human hepatoma SMMC-7721 cells. The results suggested that the present method for determination of eupatilin is simple, fast, accurate, sensitive, and reproducible. Hepatoma SMMC-7721 cells are quite sensitive to Folium artemisiae Argyi extract, which may be associated with its suppression of p53, Topo II, and bcl-2 expressions. Abbreviations used: HPLC: High-performance liquid chromatograph; OD: Optical density; RSD: Relevant standard derivation; IC50: Inhibitory 50% concentration.

Effect of NET-1 siRNA conjugated sub-micron bubble complex combined with low-frequency ultrasound exposure in gene transfection.

The present study evaluated the effect of NET-1 siRNA-conjugated sub-micron bubble (SMB) complexes combined with low-frequency ultrasound exposure in gene transfection. The NET-1 gene was highly expressed level in SMMC-7721 human hepatocellular carcinoma cell line. The cells were divided into seven groups and treated with different conditions. The groups with or without low-frequency ultrasound exposure, groups of adherent cells, and suspension cells were separated. The NET-1 siRNA-conjugated SMB complexes were made in the laboratory and tested by Zetasizer Nano ZS90 analyzer. Flow cytometry was used to estimate the transfection efficiency and cellular apoptosis. Western blot and quantitative real-time polymerase chain reaction (qPCR) were used for the estimation of the protein and mRNA expressions, respectively. Transwell analysis determined the migration and invasion capacities of the tumor cells. The results did not show any difference in the transfection efficiency between adherent and suspension cells. However, the NET-1 siRNA-SMB complexes combined with low-frequency ultrasound exposure could enhance the gene transfection effectively. In summary, the NET-1 siRNA-SMB complexes appeared to be promising gene vehicle.

Induction of apoptosis by an oleanolic acid derivative in SMMC-7721 human hepatocellular carcinoma cells is associated with mitochondrial dysfunction.

The aim of the present study was to investigate the effects of an oleanolic acid derivative, a novel antitumor drug, on the growth of SMMC-7721 human hepatocellular carcinoma cells and the underlying mechanism. An MTT assay was performed to determine the cytotoxicity of the oleanolic acid derivative. Cell membrane integrity was assessed using fluorescence microscopy to assess the uptake of annexin V-FITC/propidium iodide (PI). Western blotting was used to detect the apoptosis-associated proteins B cell lymphoma-2 (Bcl-2), Bax, caspase-9 and caspase-3. A spectrophotometer was used to analyze the intracellular adenosine triphosphate (ATP) expression level. The loss of mitochondrial membrane potential was detected by performing the JC-1 assay. ELISA was used to evaluate the content of cytochrome c (Cyt-C). The oleanolic acid derivative reduced the cell viability of SMMC-7721 cells in a dose- and time-dependent manner. The half maximal inhibitory concentration values of the oleanolic acid derivative in SMMC-7721 cells at 24, 48 and 72 h were 26.80, 11.85, and 6.66 µM, respectively. The antiapoptotic-protein Bcl-2 was downregulated, and the proapoptotic protein Bax was upregulated following treatment with the oleanolic acid derivative for 48 h. The oleanolic acid derivative induced the cleavage of caspase-9 and caspase-3 as well as promoted annexin V-FITC/PI uptake in SMMC-7721 cells. Furthermore, treatment of SMMC-7721 cells with the oleanolic acid derivative induced a reduction of the intracellular ATP expression level, loss of ΔΨm and Cyt-C release from the mitochondria. The oleanolic acid derivative induced apoptosis in SMMC-7721 human cells. Mitochondrial dysfunction was involved in the anticancer effects of this derivative on SMMC-7721 human cells.

Berbamine induces SMMC-7721 cell apoptosis via upregulating p53, downregulating survivin expression and activating mitochondria signaling pathway.

Hepatocellular carcinoma (HCC) is a primary malignancy in the liver, which is a global health problem. The present study aimed to observe the apoptotic effects of berbamine on SMMC-7721 cell lines and to investigate the effects of berbamine on induction of the intrinsic apoptotic pathway. The human HCC SMMC-7721 cells were cultured and cell morphology observed using a phase contrast microscope. SMMC-7721 cell apoptosis was examined by employing a flow cytometry assay. The nuclei of SMMC-7721 cells were stained with DAPI and observed by utilizing a laser fluorescence microscope. Cytochrome c (Cyto c) levels were evaluated by using immunofluorescence staining. The reverse transcription-semi-quantitative polymerase chain reaction (RT-sqPCR) and western blot analysis were used to examine the mRNA and protein levels of B-cell lymphoma 2, (Bcl-2), Bax, Bcl-2-associated X, apoptosis regulator, p53 and survivin, respectively. Berbamine inhibited SMMC-7721 cell growth at 20 and 0 µmol/l, compared with control group (0 µmol/l berbamine). DAPI results demonstrated that berbamine affected the nucleus morphology of SMMC-7721 cells. Berbamine at a concentration of 20 µmol/l (P<0.05) and 40 µmol/l (P<0.01) significantly enhanced apoptosis rate compared with control group. Berbamine triggered Cyto c release from SMMC-7721 cell nuclei to the cytoplasm. Berbamine (10, 20, 40 µmol/l) significantly enhanced Bax and p53 levels and decreased Bcl-2 and survivin levels compared with control group, according to RT-sqPCR and western blot assay findings. In conclusion, berbamine induced SMMC-7721 cell apoptosis, through upregulating p53 expression and downregulating survivin expression, which further triggered mitochondria signaling pathway-mediated apoptosis.

Shikonin causes apoptosis by disrupting intracellular calcium homeostasis and mitochondrial function in human hepatoma cells.

Shikonin is known to suppress proliferation and induce apoptosis in a variety of cancer cell lines. In the present study, SMMC-7721 human hepatocellular carcinoma cells were treated with shikonin (1, 2 or 4 µM) for 12–48 h. Cell morphological alterations and DNA damage were determined. Furthermore, changes in cell cycle, mitochondrial transmembrane potential, calcium homeostasis and levels of reactive oxygen species were measured. Shikonin-treated SMMC-7721 cells exhibited morphological changes and DNA damage. Shikonin inhibited cell proliferation causing cell cycle arrest at the G0/G1 phase and induced apoptosis in a dose- and time-dependent manner. Shikonin-induced apoptosis was associated with activation of caspases-3, −8 and −9, elevated levels of intracellular Ca2+ and reactive oxygen species, reduced mitochondrial membrane potential and enhanced efflux of Ca2+ and K+. Gene expression B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax), p53 and caspase-3 was up-regulated, whereas Bcl-2 expression was downregulated. Shikonin caused apoptosis by inhibiting cell cycle progression, disrupting Ca2+ homeostasis, inducing oxidative stress and triggering mitochondrial dysfunction. Activation of caspases-3, −8 and −9, K+ efflux, and regulation of Bax, Bcl-2, p53 and caspase-3 expression are involved in the process. These results provide in-depth insight into the mechanisms of action of shikonin in the treatment of cancer.