Human SMMC-7721 Cells Research Articles

Proteomics and Metabolomics Analysis Reveals the Toxicity of ZnO Quantum Dots on Human SMMC-7721 Cells

ZnO quantum dots (QDs) are composed of less toxic metals than other QDs but have the same interesting photochemical properties. Thus, they have received considerable attention recently. Nevertheless, their toxicity cannot be ignored. In this study, we incubated ZnO QDs with human SMMC-7721 cells for 24 h to assess their nanotoxicity through proteomics (Fold change >1.5 and p-value <0.05) and metabolomics (Fold change ≥ 1.5; VIP ≥ 1; p-value < 0.05) analyses. Both of 174 and 219 significantly changed metabolites were identified in human SMMC-7721 cells treated with 20 and 50 µg/mL ZnO QDs, respectively. ZnO QDs significantly modified metabolic pathways, including purine metabolism, ferroptosis, morphine addiction, alcoholism, cGMP-PKG signaling, and Cushing syndrome. Moreover, we identified 105 and 8 differentially expressed proteins in cells treated with 20 and 50 µg/mL ZnO QDs, and the pathways of alcoholism and Cushing syndrome were enriched. ZnO QDs did not affect cell viability in a CCK8 assay, but disturbed the level of intracellular metabolites and proteins at 20 µg/mL. The KEGG analyses of the metabolomics and proteomics data both enriched the alcoholism and Cushing syndrome pathways. These results provide an experimental basis for future research on the safe use of nanomaterials.

Comprehensive lipidomics in apoM-/- mice reveals an overall state of metabolic distress and attenuated hepatic lipid secretion into the circulation

Apolipoprotein M (apoM) participates in both high-density lipoprotein and cholesterol metabolism. Little is known about how apoM affects lipid composition of the liver and serum. In this study, we systemically investigated the effects of apoM on liver and plasma lipidomes and how apoM participates in lipid cycling, via apoM knockout in mice and the human SMMC-7721 cell line. We used integrated mass spectrometry-based lipidomics approaches to semiquantify more than 600 lipid species from various lipid classes, which include free fatty acids, glycerolipids, phospholipids, sphingolipids, glycosphingolipids, cholesterol, and cholesteryl esters (CEs), in apoM-/- mouse. Hepatic accumulation of neutral lipids, including CEs, triacylglycerols, and diacylglycerols, was observed in apoM-/- mice; while serum lipidomic analyses showed that, in contrast to the liver, the overall levels of CEs and saturated/monounsaturated fatty acids were markedly diminished. Furthermore, the level of ApoB-100 was dramatically increased in the liver, whereas significant reductions in both ApoB-100 and low-density lipoprotein (LDL) cholesterol were observed in the serum of apoM-/- mice, which indicated attenuated hepatic LDL secretion into the circulation. Lipid profiles and proinflammatory cytokine levels indicated that apoM-/- leads to hepatic steatosis and an overall state of metabolic distress. Taken together, these results revealed that apoM knockout leads to hepatic steatosis, impaired lipid secretion, and an overall state of metabolic distress.

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

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.

Oleanolic acid derivatives inhibit the Wnt/β-catenin signaling pathway by promoting the phosphorylation of β-catenin in human SMMC-7721 cells.

Oleanolic acid, isolated from privet, has shown antitumor effects in several cancers. However, the underlying molecular mechanism associated with these effects is largely unknown. In this study, we explored the effect of oleanolic acid derivatives on the Wnt/β-catenin signaling pathway in human hepatocellular carcinoma SMMC-7721 cells. The mRNA and protein levels of related genes were determined by real-time quantitative PCR and Western blot, respectively. Treatment of SMMC-7721 cells with oleanolic acid derivatives led to the downregulation of the mRNA and protein levels of β-catenin, c-myc, and cyclin D1. Treatment with oleanolic acid derivatives decreased the levels of β-catenin in both the cytoplasm and the nucleus. Moreover, oleanolic acid derivatives promoted the phosphorylation of β-catenin (Ser33/37/Thr41) in the cytoplasm. Our results suggest that oleanolic acid derivatives inhibit the Wnt/β-catenin signaling pathway by stimulating the phosphorylation of β-catenin (Ser33/37/Thr41) in human SMMC-7721 cells.

Meloxicam combined with sorafenib synergistically inhibits tumor growth of human hepatocellular carcinoma cells via ER stress-related apoptosis

Sorafenib (SOR) is a promising treatment for advanced hepatocellular carcinoma (HCC). However, the precise mechanisms of toxicity and drug resistance have not been fully explored and new strategies are urgently needed for HCC therapy. Meloxicam (MEL) is a selective cyclooxygenase-2 (COX-2) inhibitor which elicits antitumor effects in human HCC cells. In the present study, we investigated the interaction between MEL and SOR in human SMMC‑7721cells and the role endoplasmic reticulum (ER) stress exerts in the combination of SOR with MEL treatment-induced cytotoxicity. Our results revealed that the combination treatment synergistically inhibited cell proliferation and enhanced apoptosis. Furthermore, the combination treatment enhanced ER stress-related molecules which involved in SMMC-7721 cell apoptosis. GRP78 knockdown by siRNA or co-treatment with MG132 significantly increased this combination treatment-induced apoptosis. In addition, we found that the combination treatment suppressed tumor growth by way of activation of ER stress in invivo models. We concluded that the combination of SOR with MEL treatment-induced ER stress, and eventually apoptosis in human SMMC-7721cells. Knockdown of GRP78 using siRNA or proteosome inhibitor enhanced the cytotoxicity of the combination of SOR with MEL-treatment in SMMC-7721cells. These findings provided a new potential treatment strategy against HCC.

Effect of oleic acid on the levels of eight metal ions in human hepatoma SMMC-7721 cells.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Its incidence is rising worldwide. However, no specific therapy has been shown to be effective in its treatment. In the present study, the in vitro NAFLD model was established in human SMMC-7721 cells by using oleic acid (OA). Then, content changes of eight cations, including sodium, magnesium, potassium, calcium, iron, copper, zinc, and manganese, were investigated in the experimental model. The results showed that OA induced a decrease in magnesium level, while an increase in iron one. Additionally, the supplementation of magnesium in the cell culture model was studied. It showed that magnesium ameliorated lipid accumulation induced by OA. Our results suggest that magnesium could decrease the risk of NAFLD and be used as a promising candidate for the treatment of NAFLD.

Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways

BackgroundLanthanide cerium oxide (CeO2) nanoparticles have extensive applications in industrial fields, and concerns regarding their potential toxicity in humans and their environmental impact have increased. We investigated the underlying molecular mechanisms by which CeO2 nanoparticles induce toxicity in human hepatoma SMMC-7721 cells. ResultsOur results demonstrated that CeO2 nanoparticles reduced viability, caused dramatic morphological damage, and induced apoptosis in SMMC-7721 cells. CeO2 nanoparticles significantly increased the production of reactive oxygen species (ROS) and malondialdehyde (MDA), and significantly reduced the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT). The phosphorylation levels of ERK1/2, JNK and p38 MAPK were significantly elevated after treatment with CeO2 nanoparticles. Pretreatment with the antioxidant N-acetyl-cysteine (NAC): reduced the induction of ROS and MDA by CeO2 nanoparticles; recovered the activity of SOD, GSH-px and CAT; reduced the phosphorylation levels of ERK1/2, JNK and p38; and attenuated CeO2 nanoparticles-induced damage and apoptosis in SMMC-7721 cells. ConclusionsOur data demonstrated that CeO2 nanoparticles induced damage and apoptosis in human SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways.

Inhibitory effect of M007-mediated photodynamic therapy on proliferation of human hepatoma SMMC-7721 cells

Objective To investigate the effect of new photosensitizer M007-mediated photodynamic therapy on human hepatoma SMMC-7721 cells in vitro and proliferation capacity of the residual cells. Methods Subcultured SMMC-7721 human hepatocellular carcinoma cells lines, methylene blue derivatives M007 as the photosensitizer, setting different concentrations of M007-PDT groups （2,4,16 μmol/L）, multi-photo source system illuminates 1 x 106 cells in 630-660 nm wave length for 10 min （luminous power density is 0. 036 W/cm2 ,the light dosage is 2. 16 J/cm2） ,analyzing cell mortality and manner of death by flow cytometry, Methyl thiazol tetrazolium （MTT） test and colony formation assay to assess residual cell proliferation. Results When the M007 concentration in 2,4,16 μmol/L, the cell mortality in M007-PDT groups were （ 68.50±3.33 ） %, （96. 70±2. 63 ） %, （99. 30±0. 30） % respectively; in 2,4 and 6μmol/L M007-PDTgroups, residual cell growth curve is low and flat, and the optical density value is not increased with the incubation time （P 〉 0.05 ） ,4,16μmol/L M007-PDT groups failed to form a colony, but 2μmol/L M007- PDT group had a few colony formation occasionally. Conclusion M007 doses in 4,16 μmol/L,M007 mediated photodynamic therapy on SMMC-7721 cell line has the exactly inactivated effect, and the residual cells lost long-term proliferative capacity in vitro. Key words: Carcinoma, hepatocellular; Photochemical therapy; Proliferation

The research of compound matrine injection inducing human hepatoma SMMC-7221 cell to apoptosis

Objective To explore the compound matrine injection on human hepatoma smmc-7721 cells' proliferation.Methods Human hepatoma smmc-7721 cells were sterilely cultured.Assayed the effects of compound matrine injection on smmc-7721 cells by MTT.The concentrations of the compound matrine injection were 3.34,2.38,1.43 and 0.48 mg/ml; Used the double staining method of annexin V-fluorescein isothiocyanate(Annexin V-FITC)/propidium iodide (propidium iodide,PI) to measure the apoptosis of hepatoma cell.The concentrations of the compound matrine injection were 0.86,1.72,and 3.43 mg/ml.Results The apoptosis rates of compound matrine injection with different concentration of 3.34,2.38,1.43 and 0.48 mg/ml on liver cancer cell SMMC-7721 were51.03％、53.67％、49.83％ and 0.03％ respectively after 24 hours; and 67.14％、65.35％、62.25％ and 0.05％ respectively after 48 hours;and 74.16％、68.77％、66.04％ and 26.02％respectively after 72 hours.The apoptosis rates of compound matrine injection with different concentration of 0.86,1.72 and 3.43 mg/ml on liver cancer cell SMMC-7721 were(2.86±0.35)％,(15.16± 1.15)％and(13.17±0.40)％ respectively after 24 hours; and (8.57±0.44)％,(28.07±0.76)％ and (29.81±8.10)％respectively after 48 hours,which were significantly higher than the control group (1.05±0.09)％(P＜0.05).Conclusion The compound matrine injection has a significant inhibition on human hepatoma smmc-7721 cells' proliferation and the mechanism was its inducing cell apoptosis. Key words: Compound matrine injection; smmc-7721 cells; Cell apoptosis

Mechanism of apoptotosis induced by ortho-topolin riboside in human hepatoma cell line SMMC-7721

The naturally occurring cytokinin, ortho-topolin riboside (oTR), has been recently reported to have a strong anticancer effect. However, the molecular mechanism has not been elucidated. From our research we found that oTR strongly inhibited the proliferation of SMMC-7721 cells inducing apoptosis. After oTR treatment, up-regulation of the protein levels of pro-apoptotic Bax and the down-regulation of the anti-apoptotic proteins, Bcl-2 and Bcl-xL was observed, leading to the loss of mitochondrial membrane potential, the release of cytochrome c from the mitochondria into the cytosol, the downstream activation of caspase-9 and caspase-3, as well as the cleavage of poly ADP-ribose-polymerase (PARP), the effect of apoptosis could be blocked by the pan-specific caspase inhibitor z-VAD-fmk and caspase-9-specific inhibitor z-LEHD-fmk. Moreover, oTR was shown to inhibit the activation of the extracellular signal-regulated kinase-1/2 (ERK1/2) as well as the Akt pathway. These results suggest that oTR interferes with the mitogen-activated protein kinase (MAPK) and Akt pathways and induces the apoptosis of human SMMC-7721 cells through the activation of intrinsic mitochondria-mediated pathways. However, the apoptosis was completely prevented when cells were treated with A-134974, an inhibitor of adenosine kinase, it indicated that the intracellular phosphorylation of oTR is necessary for its cytotoxic effects to SMMC-7721 cells.

βig‐h3 regulates store‐operated Ca2+ entry and promotes the invasion of human hepatocellular carcinoma cells

βig-h3 is a TGF-β (transforming growth factor β)-induced ECM (extracellular matrix) protein that induces the secretion of MMPs (matrix metalloproteinases). However, the mechanism of induction is yet to be established. In this study, siRNAs (small interfering RNAs) targeted against βig-h3 were transfected into SMMC-7721 cells [a HCC (human hepatocellular carcinoma) cell line] to knockdown the expression of βig-h3. We found that NiCl2, a potent blocker of extracellular Ca2+ entry, reduced βig-h3-induced secretion of MMP-2 and -9. Further investigation suggested that reduction in the levels of βig-h3 decreased the secretion of MMP-2 and -9 that was enhanced by an increase in the concentration of extracellular Ca2+. SNAP (S-nitroso-N-acetylpenicillamine), a NO (nitric oxide) donor, and 8-Br-cGMP (8-bromo-cGMP) inhibited thapsigargin-induced Ca2+ entry and MMP secretion in the invasive potential of human SMMC-7721 cells. Further, the inhibitory effects of 8-Br-cGMP and SNAP could be significantly enhanced by down-regulating βig-h3. βig-h3 attenuates the negative regulation of NO/cGMP-sensitive store-operated Ca2+ entry. Our findings suggest that the expression of βig-h3 might play an important role in the regulation of store-operated Ca2+ entry to increase the invasive potential of HCC cells.

Effects of HBV-X on the malignant phenotypes of human hepatocellular carcinoma cell line SMMC-7721

Objective To study the effects of HBV-X on the malignant phenotypes of human carcinoma cell line SMMC-7721. Methods Human hepatocellular carcinoma SMMC-7721 cells were transfect-ed with plasmid pcDNA3. 1(-)-X and cells transfected with a mock plasmid served as controls. HBV-X expression was detected by using reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting, and concentrations of HBV-X, MMP-2, MMP-9 and urinase plasminogen activator (uPA) were measured by enzyme-linked immunosorbent assay (ELISA). A series of functional assays in vitro were used to monitor the changes of SMMC-7721 malignant phenotypes. Results HBV-X expression of SMMC-7721 cells was elevated after transfection. Concentrations of HBV-X, MMP-2, MMP-9 and uPA in the medium of SMMC-7721 cells after transfection were higher than those of the controls [(13.02 ± 2. 12)μg/L vs (4. 07 ±0.37)μg/L, (49.04±4.07)μg/L vs (25.15 ±5.43)μg/L, (27. 82 ±2. 25)μg/L vs (7.89± 1.27)μg/L, (10.78±3.23)μg/L vs (1.24±0.34)μg/L respectively,P<0.01]. Functional assays ire vitro indicated that SMMC-7721 cells after transfection showed higher adhensive, migrant and invasive capabilities than those of the controls [cell adhesion rate: (85.33±14.78) % vs (57.34±2.52)% ; number of outer surface cells in migrant assay: 24.3±1.9 vs 12.3±2.5; cell number in the invasive assay: 18.2± 3.2 vs 9.3±2.3 respectively.P<0.05]. Conclusion HBV-X might enhance the expression of MMP-2, MMP-9 and uPA to promote malignant phenotypes of SMMC-7721 cells. Key words: Carcinoma, hepatocellular; HBV-X protein

Selective inhibitory effects of artemisinin and holotransferrin on human hepatocellular carcinoma SMMC-7721 cells

Objective To investigate the selective inhibitory effect of artemisinin and holotransferrin on human hepatoeellular carcinoma SMMC-7721 cells in vitro.Methods MTT and trypan blue assay were used to assess the proliferation and viability of SMMC-7721 eeUs treated with artemisinin and holotransferrin.The inhibition of colony formation elicited by artemisinin was determined by colony formation assay.Results Artemisinin significantly inhibited SMMC-7721 cell growth,colony formation and cell viability at various concentrations (P < 0.05).It was found that combined incubation with holotransferrin, which increased the concentration of ferrous iron in cancer cells, sensitized the growth inhibitory effect of artemisinin on SMMC-7721 cells (P < 0.01).Conclusion Artemisinin and holotransferrin effectively inhibited SMMC-7721 cell viability, growth, and proliferation, which may provide a potential therapeutic choice for liver cancer. Key words: Carcinoma, hepatoeellular; Artemisinin; Transferrin; Cell growth; Cell via-bility

HGF upregulates Mcl-1 expression through PDK/AKT pathways in SMMC7721 cells

Objective To investigate the role of MAPK, PI3K/AKT pathways in HGF-induced Mcl-1 upregulation in SMMC-7721 cells. Methods The expression of c-met was detected by RT-PCR. SMMC-7721 cells were exposed to HGF, phospborylation of MAPK, Akt and Mcl-1 expression was detected by Western blot. Results The SMMC-7721 expressed c-met, HGF stimulates phospho-ralation of MAPK, PI3K/AKT at 5min after adding HGF and achieves the peak at 2h. Furthermore, HGF up-regulates Mcl-1 expression was in a time- and concentration-dependent manner in human SMMC7721 cell lines. The upregulation of Mcl-1 induced by HGF was inhibited when PI3k pathway was abolished by the specific inhibitor Wortmannin. However, it was not inhibited when MAPK path-way was abolished by inhibitor PD98059. Conclusion HGF can activate both the PI3K and MAPK pathways and HGF upregulates Mcl-1 expression via PI3K/AKT pathway. Key words: HGF; SMMC-7721 cells; Mcl-1; PI3K/AKT

Group I but not Group II NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus (NPV) is divided into Group I and Group II based on the phylogenetic analysis. It has been reported that Group I NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group II NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group II had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.