Human Hepatoma Cell Line SMMC-7721 Research Articles

A new cytochalasin from endophytic Phomopsis sp. IFB-E060

AimTo study the chemical constituents of the solid culture of the endophyte Phomopsis sp. IFB-E060 in Vatica mangachapoi. MethodIsolation and purification were performed through silica gel column chromatography, gel filtration over Sephadex LH-20, ODS column chromatography, and HPLC. Structures of the isolated compounds were elucidated by a combination of spectroscopic analyses (UV, CD, IR, MS, 1D, and 2D NMR). The cytotoxicity of the isolates was evaluated in vitro by the MTT method against the human hepatocarcinoma cell line SMMC-7721. ResultsFive compounds were isolated from the solid culture of the endophyte Phomopsis sp. IFB-E060 and their structures were identified as 18-methoxy cytochalasin J (1), cytochalasin H (2), (22E, 24S)-cerevisterol (3), ergosterol (4), and nicotinic acid (5). Compound 1 had an inhibition rate of 24.4% at 10 μg·mL−1 and 2 had an IC50 value of 15.0 μg·mL−1, while a positive control 5-fluorouracil had an inhibition rate of 28.7% at 10 μg·mL−1. Conclusion18-Methoxy cytochalasin J (1), produced by endophytic Phomopsis sp. IFB-E060, is a new cytochalasin with weak cytotoxicity to the human hepatocarcinoma cell line SMMC-7721

Impact of Mucin1 knockdown on the phenotypic characteristics of the human hepatocellular carcinoma cell line SMMC-7721.

Mucin1 (MUC1) is a transmembrane glycoprotein that plays a key role as an oncogene in the tumorigenesis of many human adenocarcinomas. However, the role of MUC1 in human hepatocellular carcinoma (HCC) progression remains unclear. In the present study, we silenced MUC1 to investigate its effect on the human HCC cell line SMMC-7721 and found that knockdown of MUC1 significantly inhibited cell proliferation, enhanced cell-cell aggregation and induced apoptosis. No significant differences were found in invitro migration or invasion. We also observed that knockdown of MUC1 decreased the translocation of β‑catenin to the nucleus, reduced the activity of T cell factor and blocked the expression of cyclinD1 and c-Myc. In addition, MUC1 knockdown enhanced the expression of E-cadherin, a molecular chaperone of β‑catenin that plays an important role in cell-cell aggregation. Invivo assays demonstrated that there was no tumor growth in mice injected with MUC1-silenced cells. Global gene expression analysis showed that a series of genes encoding molecules in the Wnt/β‑catenin, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), insulin, transforming growth factorβ (TGF-β) and vascular endothelial growth factor (VEGF) signaling pathways were all influenced by the knockdown of MUC1, and these may contribute to the phenotypic alterations observed. Collectively, our results indicate that MUC1 plays a key role in HCC tumorigenesis.

Effect of Xiaoaiping, oxaliplatin and Endostar, alone or in combination, on cell proliferation in human hepatoma cell line SMMC-7721

Effect of Xiaoaiping, oxaliplatin and Endostar, alone or in combination, on cell proliferation in human hepatoma cell line SMMC-7721

Effect of Qishen-Qinggan decoction on proliferation and apoptosis of human hepatocellular carcinoma cellline SMMC-7721

Objective To investigate effect of Qishen-Qinggan decoction on proliferation and apoptosis of human hepatocellular carcinoma cellline SMMC-7721.Methods SMMC-7721 cells were cultivated in vitro.Logarithmic growth phase cells were divided into a drug intervention group and a control group.SMMC-7721 cells were treated with Qishen-Qinggan decoction of 0.135、0.27、0.54、1.08、2.16 g/ml respectively for 12、24、48 hours in the drug intervention group while the control group remained untreated.The inhibition rate of SMMC-7721 cells were detected by MTT assay,cell apoptotic rate were measured by flow cytometry analysis.Results Qishen-Qinggan decoction of 0.135,0.27,0.54,1.08,2.16 g/ml had a significantly inhibitory effect on SMMC-7721 cells in a dose and time dependent manner.OD values of 12 hours were 0.89±0.05,0.85±0.05,0.80±0.06,0.78± 0.02,0.69±0.07,OD values of 24 hours were 0.77±0.07,0.74±0.07,0.59±0.07,0.50±0.09,0.39±0.08,OD values of 48 hours were 0.78±0.05,0.61±0.08,0.44±0.10,0.39±0.08,0.34±0.07 respectively.Each drug intervention group had significant difference compared with control group.Qishen-Qinggan decoction of 0.27,0.54 g/ml could induce apoptosis of SMMC-7721 were 11.19 ± 2.23,15.69 ± 2.51,compared with control group of 1.41 ± 0.22.Apoptotic rates of Qishen-Qinggan decoction of 1.08 g/ml had extremely significant difference with the control group (41.83 ± 7.11 vs 1.41 ± 0.22).Conclusion Qishen-Qinggan decoction could inhibit the proliferation of SMMC-7721 cells probably by inducing cell apoptosis. Key words: Qishen-Qinggan decoction; Hepatocellular carcinoma; SMMC-7721 cells; Apoptosis

Tumor-associated hepatic stellate cells significantly promote epithelial-mesenchymal transition and migration of hepatocellular carcinoma cells

Objective To establish a method for isolating and purifying tumor-associated hepatic stellate cells(tHSCs) from human hepatocellular carcinoma(HCC) tissues and to study their impact on the biological behaviors of HCC cells in vitro. Methods The tHSCs were isolated from the fresh human HCC tissue by collagenase/pronase digestion,followed by density gradient centrifugation using Nycodenze.The viability of the isolated cells was determined by trypan blue staining assay.The tHSCs were identified by immunocytoehemical staining ofα-SMA,a specific marker for activated hepatic stellate cells.The morphologic changes of the tHSCs were observed under microscope.The conditioned medium(CM) of tHSCs cultured in serum-free DMEM was collected,and then used for co-culture with HCC cell lines QGY-7701,BEL-7402,and SMMC-7721. CM of human hepatic stellate cell line LX-2 was used as control.Cell viability,proliferation,migration potential and EMTrelated protein expression changes were assessed by using the MTT assay,wound migration assay and Western blotting analysis,respectively.Results The viability of isolated tHSCs was over 95%,with a purity about 100%,and the isolated tHSCs could be passaged and cryopreserved.tHSCs significantly induced typical EMT-like morphological changes and altered expression of molecular markers in the three HCC cell lines,with the epithelial marker E-cadherin significantly decreased(P 0.05) and the stromal marker Vimentin significantly increased(P 0.05).Meanwhile,tHSCs efficiently enhanced the proliferation and migration of HCC cells in vitro.Conclusion We have established a method for isolating and purifying tHSCs from human HCC tissues,which can help future study on tHSCs.tHSCs can enhance the proliferation and migration of HCC cells through inducing EMT-like phenotype.

A standardized extract from Paeonia lactiflora and Astragalus membranaceus induces apoptosis and inhibits the proliferation, migration and invasion of human hepatoma cell lines

Paeonia lactiflora and Astragalus membranaceus are two traditional Chinese medicines, which are commonly used in Chinese herb prescription to treat liver diseases. The protective effects of the extract prepared from the roots of Paeonia lactiflora and Astragalus membranaceus (PAE) on liver fibrosis have been demonstrated in previous studies. However, its effect on hepatocellular carcinoma (HCC) has not been investigated to date. In this study, the effects of PAE on the apoptosis, proliferation, migration and invasion of the human hepatoma cell lines HepG2 and SMMC-7721 were investigated. Our data demonstrated that treatment with PAE (50-200 mg/l) caused an inhibitory effect on the proliferation of the hepatoma cell lines HepG2 and SMMC-7721. Furthermore, PAE induced apoptosis of HepG2 cells and SMMC-7721 cells, which was demonstrated by PI staining. In addition, immunocytochemistry and western blotting showed that PAE significantly decreased the expression of Bcl-2, while the expression of Bax and cleaved caspase-3 in HepG2 cells and SMMC-7721 cells was significantly increased after treatment with PAE. These results clearly demonstrated that PAE induced hepatoma cell apoptosis through increasing the Bax-to-Bcl-2 ratio and upregulating the activation of caspase-3. In addition, the results of wound healing assay and Matrigel invasion assay showed that PAE displayed inhibitory activity on the migration and invasion of HCC cells. Taken together, the present data provides evidence that PAE is a potent antineoplastic drug candidate for the treatment of HCC.

8-bromo-5-hydroxy-7-methoxychrysin targeting for inhibition of the properties of liver cancer stem cells by modulation of Twist signaling

Emerging evidence has suggested that cancer stem cells with expression of surface biomarkers including CD133 and CD44 have more aggressive biological behavior, including epithelial-mesenchymal transition (EMT), which are closely related to invasion. The upregulation and nuclear relocation of the EMT regulator Twist1 have been implicated in the tumor invasion and metastasis of human hepatocellular carcinoma (HCC). In this study, we aimed to isolate and characterize a small population of CD133+ cells that existed in the HCC cell line SMMC-7721 by MACS and investigated the possible roles of 8-bromo-7-methoxychrysin (BrMC), a synthetic analogue of chrysin, in inhibiting the properties of CD133+ sphere-forming cells (SFCs) derived from the HCC cell line SMMC-7721, namely liver cancer stem cells (LCSCs). Based on the data, BrMC inhibited the proliferation, self-renewal and invasion of LCSCs in vitro and in vivo, downregulated the expression of the LCSC biomarkers CD133 and CD44 and induced EMT by downregulating the expression of Twist and β-catenin in LCSCs. BrMC potentiated the inhibition of LCSCs self-renewal after reduction of twist protein levels, which was attenuated when twist was overexpressed. This study not only provides an important experimental and theoretical basis for investigation of BrMC in LCSCs, but also helps in the development of effective therapeutic medicine for HCC.

P21-activated kinase 5 plays essential roles in the proliferation and tumorigenicity of human hepatocellular carcinoma

To investigate the roles of P21-activated kinase 5 (PAK5) in proliferation and tumorigenicity of human hepatocellular carcinoma (HCC). HCC and matched paraneoplastictis tissue samples were obtained from 30 patients. Human HCC cell lines SMMC7721, HepG2, Hep3B, SK-HEP-1, Huh-7, and liver cell line HL-7702 were examined. The expression of PAK5 gene was studied using real-time qPCR and Western blotting. Cell proliferation was quantified with the MTT assay. Cell cycle was analyzed with flow cytometry. The tumorigenicity of Lv-shRNA-transfected HepG2 cells was evaluated in BALB/cA nude mice. The mRNA level of PAK5 was significantly higher in 25 out of 30 HCC samples compared to the matched paraneoplastic tissues. The HCC cell lines showed varying expression of PAK5 protein, and the highest level was found in the HepG2 cells. PAK5 gene silencing in HepG2 cells markedly reduced the cell proliferation and colony formation, and induced cell cycle arrest in the G1 phase. Furthermore, PAK5 gene silencing suppressed the tumor formation in nude mice, and significantly decreased the expression of HCC-related genes Cyclin D1 and beta-catenin. PAK5 may play essential roles in the initiation and progression of human HCC. Thus, it may be an effective therapeutic target or perhaps serve as a clinical diagnostic or prognostic marker in human HCC.

Inhibition of Growth and Induction of Differentiation of SMMC-7721 Human Hepatocellular Carcinoma Cells by Oncostatin M

Oncostatin M (OSM) is a multifunctional cellular regulator acting on a wide variety of cells, which has potential roles in the regulation of gene activation, cell survival, proliferation and differentiation. Previous studies have shown that OSM can induce morphological and/or functional differentiation and maturation of many tumor cells. However, the action of OSM on the induction of differentiation of human hepatocellular carcinoma (HCC) has not been reported. Here, we investigated the effects of different concentrations of OSM on human HCC cell line SMMC-7721 growth, proliferation, cell cycling, apoptosis and differentiation in vitro. Cell growth was determined via MTT assay, proliferation by cell cycle analysis, apoptosis by flow cytometry, morphology by transmission electronic microscopy, and cell function by detection of biochemical markers. Our results demonstrated that OSM strongly inhibited the growth of SMMC-7721 cells in a dose-dependent manner, associated with decreased clonogenicity. Cell cycle analysis revealed a decreased proportion of cells in S phase, with arrest at G0/G1. The apotosis rate was increased after OSM treatment compared to the control. These changes were associated with striking changes in cellular morphology, toward a more mature hepatic phenotype, accompanied by significant reduction of the expression of AFP and specific activity of γ-GT, with remarkable increase in secretion of albumin and ALP activity. Taken together, our findings indicate that OSM could induce the differentiation and reduce cell viability of SMMC-7721 cells, suggesting that differentiation therapy with OSM offers the opportunity for therapeutic intervention in HCC.

Wogonin induced cytotoxicity in human hepatocellular carcinoma cells by activation of unfolded protein response and inactivation of AKT

To investigate the potential anticancer effects of the natural flavonoid wogonin on human hepatocellular carcinoma (HCC) cells and tumor xenografts and the contribution of the unfolded protein response (UPR) and AKT pathways to the cytotoxicity of wogonin. The HCC cell lines HepG2, SMMC-7721 and Hep3B were treated with wogonin. 3-(4 5-Dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide assays were used to evaluate the cell viability. Flow cytometry assays were used to identify the cell death types and measure the concentrations of intracellular H2 O2 and Ca(2+) . Western blotting assays were used to detect the protein expression levels of members in the UPR and AKT pathways. Relative quantitative real-time polymerase chain reaction assays were used to analysis the mRNA expression levels of chop and trb3. Furthermore, the male BALB/c nude mice with SMMC-7721 xenografts were treated with wogonin. The tumor volume, tumor weight and bodyweight were monitored during the tumorigenicity assays. Wogonin significantly inhibited the viability of HCC cells by inducing apoptosis and necrosis. This cytotoxicity was at least partially attributed to the activation of the UPR pathway and consequent inactivation of AKT signaling, which resulted from the production of intracellular H2 O2 and causal release of endoplasmic reticulum Ca(2+) . Moreover, wogonin evidently repressed the growth of xenografts but slightly influenced the bodyweight of mice. Wogonin is a prospect for improving the systemic chemotherapy strategy on HCC by concurrently rectifying the aberrant UPR and AKT signaling pathways, which are crucial to the biology of HCC.

Parasporal Crystal Proteins of <i>Bacillus thuringiensis</i> ly36 Strain Demonstrating Bacteriacidal and Cancer Cell-Killing Activities

The growth of Bacillus subtilis was highly inhibited by crystal proteins of Bacillus thuringiensis ly36 strain with or without the treatment of proteinase K. And human hepatoma cell lines SMMC-7721 and Bel-7402 were killed by the proteinase K-treated crystal proteins of B. thuringiensis ly36 strain.

PH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein.

BackgroundEncapsulating exogenous proteins into a nanosized particulate system for delivery into cells is a great challenge. To address this issue, we developed a novel nanoparticle delivery method that differs from the nanoparticles reported to date because its core was composed of cross-linked dextran glassy nanoparticles which had pH in endosome-responsive environment and the protein was loaded in the core of cross-linked dextran glassy nanoparticles.MethodsIn this study, dextran in a poly(ethylene glycol) aqueous two-phase system created a different chemical environment in which proteins were encapsulated very efficiently (84.3% and 89.6% for enhanced green fluorescent protein and bovine serum albumin, respectively) by thermodynamically favored partition. The structures of the nanoparticles were confirmed by confocal laser scanning microscopy and scanning electron microscopy.ResultsThe nanoparticles had a normal size distribution and a mean diameter of 186 nm. MTT assays showed that the nanoparticles were nontoxic up to a concentration of 2000 μg/mL in human hepatocarcinoma cell line SMMC-7721, HeLa, and BRL-3A cells. Of note, confocal laser scanning microscopy studies showed that nanoparticles loaded with fluorescein isothiocyanate-bovine serum albumin were efficiently delivered and released proteins into the cytoplasm of HeLa cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that nanoparticles with a functional protein (apoptin) efficiently induced significant tumor cell apoptosis, which was confirmed by DAPI staining.ConclusionOur findings indicate that these nanoparticles meet the high demands for delivering protein medicines and have great potential in protein therapy.

General Transcription Factor IIB Overexpression and a Potential Link to Proliferation in Human Hepatocellular Carcinoma

The general transcription factor IIB (TFIIB) plays a central role in preinitiation complex (PIC) assembly, providing a bridge between promoter-bound TFIID and RNA Polymerase II (RNA POLII). TFIIB functionally counteracts the transcriptional activation of hepatitis B virus X protein (HBx), which has been shown to play a role in the development of human hepatocellular carcinoma (HCC). However, the function of TFIIB in HCC remains unclear. In this article, we demonstrate that TFIIB plays an important role in HCC pathogenesis. TFIIB expression was immunohistochemically examined in a series of 100 HCC tissue specimens. The expression level of TFIIB showed significant correlation with the histological grade (P = 0.030), the level of AFP (P = 0.011) and the proliferation marker Ki-67 (P = 0.0002). High TFIIB expression level correlated with poor survival. Western blot analysis also confirmed that the TFIIB protein was overexpressed in HCC tissue compared to benign normal tissue. Additionally, Western blot and qRT-PCR analyses showed a high expression level of TFIIB protein in the HCC cell lines SMMC7721, HepG2, BEL7404, and Huh7 and the immortalized normal line BEL7702 but a lower expression in the normal Chang hepatocyte cell line. Following the release of Huh7 cells from serum starvation, the expression of TFIIB was upregulated. A cell growth assay suggested that TFIIB was involved in the proliferation and growth of HCC cells. In conclusion, our results demonstrate that TFIIB overexpression may play essential roles in the pathogenesis of hepatocellular carcinoma by affecting the proliferation of HCC cells.

Overexpression of LSD1 in hepatocellular carcinoma: a latent target for the diagnosis and therapy of hepatoma

The aim of this study was to investigate the expression of LSD1 in human hepatocellular carcinoma (HCC) cell lines and HCC samples. In this study, we examined LSD1 expression in 60 paired liver cancer tissues and adjacent noncancerous tissues by Western blot. In addition, we analyzed LSD1 expression in 198 HCC samples by immunohistochemistry. The relationship between LSD1 expression and clinicopathological features was investigated. The HCC cell line SMMC-7721 was transfected with LSD1 siRNA expressing plasmids. We subsequently examined in vitro cell growth using the MTT assay and anchorage-independent growth through a soft-agar colony-formation assay. In addition, the expression levels of Bcl-2 and c-Myc were also examined. Immunohistochemistry and Western blotting consistently confirmed LSD1 overexpression in HCC tissues compared with adjacent non-neoplastic tissues (P < 0.01). Additionally, immunostaining showed more LSD1-positive cells in the higher tumor stage (T3-4) and tumor grade (G3) than in the lower tumor stage (T1-2, P < 0.001) and tumor grade (G1-2, P < 0.001). Knockdown of LSD1 expression in HCC cells led to decreased cell proliferation. The expression of Bcl-2 and c-Myc were down-regulated after transfection of LSD1 siRNA into HCC cell line SMMC-7721. In conclusion, because LSD1 was overexpressed in HCC and has an important role in the development of HCC, LSD1 could be a latent target in the diagnosis and therapy of HCC.

MCM7 expression predicts post‐operative prognosis for hepatocellular carcinoma

Dysregulation of minichromosome maintenance protein 7 (MCM7) was previously identified in multiple human malignancies. The clinical significance of MCM7 expression is yet to be delineated in patients with hepatocellular carcinoma (HCC). Paired cancerous and non-cancerous specimens from 87 patients with HCC who underwent resection were used for the immunohistochemical evaluation of MCM7 expression. Effect of sorafenib on the expression of MCM7 was tested in two human HCC cell lines SMMC-7721 and PLC/PRF/5. Non-cancerous tissues were negative for immunohistochemical staining for MCM7 expression. Nuclear MCM7 was expressed in 42 of 87 HCC (48.2%) and was correlated with hepatitis B virus infection (P = 0.020), intrahepatic metastasis (P = 0.022) and vascular invasion (P = 0.013). Moreover, its expression was correlated with shorter overall survival (P = 0.033). Multivariate analysis showed that MCM7 expression was an independent prognostic factor for overall survival(P = 0.041). Sorafenib inhibited the expression of MCM7 in a concentration-dependent manner in vitro. The current findings suggested that MCM7 expression may be a useful predictor of prognosis in patients with HCC after resection. Adjuvant therapy with sorafenib might be a valuable therapeutic strategy for MCM7-positive HCC patients.

Synthesis and characterization of cisplatin‐loaded, EGFR‐targeted biopolymer and in vitro evaluation for targeted delivery

The design of smart targeted drug delivery systems that deliver drugs to specific cancer cells will give rise to cancer treatments with better efficacy and lower toxicity levels. We report the development and characterizations of maleimide-functionalized biopolymer (Mal-PGA-Asp) as an effective targeted drug delivery carrier synthesized from an amidation reaction between aspartylated PGA (PGA-Asp) and N-(maleimidohexanoyl)-ethylenediamine (NME). The epidermal growth factor receptor (EGFR) targeting peptide (TP13) was conjugated to Mal-PGA-Asp to obtain the targeting carrier (TP13-Mal-PGA-Asp). Cisplatin was finally loaded by complexation to form a biocompatible and tumor targeted therapeutic drug (TP13-Mal-PGA-Asp3-Pt). The resultant biopolymer with an average size 87 ± 28 nm showed a sustainable release profile with a half-maximal release time (t(1/2)) of approximately 15 h in physiological saline. Fluorescence imaging and flow cytometry analysis revealed that TP13 significantly enhanced the cellular uptake of TP13-Mal-PGA-Asp3-Pt in the human hepatoma cell line SMMC-7721. The IC(50) value demonstrated the superior anticancer activity of TP13-Mal-PGA-Asp3-Pt over PGA-Asp-Pt. Therefore, the newly developed drug carrier (TP13-Mal-PGA-Asp) obtained in this study may provide an efficient and targeted delivery of anticancer drugs, presenting a promising targeted chemotherapy in EGFR-positive cancers.

Inhibitory effect of vitamin K2 and phosphatidylcholine on hepatocellular carcinoma cells

Objective To investigate the synergistic effect and mechanism of vitamin K2 and phosphatidylcholine (PC) on hepatocellular carcinoma (HCC) cells.Methods Synergism between vitamin K2 ( 1.25-500.O0 μ moI/L) and PC ( 1.25-400.00 μmoI/L) was studied in human hepatoma cell line SMMC-7721 in vitro with cell counting Kit-8 ( CCK-8 ) kit and Annexin V-FITC/PI kit.The key proteins of Caspase apoptosis pathway were detected by using Western blotting.Results The combined administration of vitamin K2 (500.00 μmol/L) and PC (20.00 μmol/L) induced 65.1％ apoptosis rate of HCC cells at 72nd h.Western blotting revealed the significant difference in the differential expression of procaspase-3 and procaspase-9 in the experimental group ( P ＜ 0.05 ),while procaspase-8 changed unsignificantly ( P ＞0.05 ).Conclusion Vitamin K2 and PC exhibited synergistic effects against hepatocarcinogenesis via activating Caspase-9. Key words: Carcinoma, hepatoma; Vitamin K2; Phosphatidylcholine

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.

Phloretin induced apoptosis of human hepatoma cells SMMC-7721 and its correlative biological mechanisms

In order to investigate the proliferative and apoptotic effects of phloretin on human hepatoma cell line SMMC-7721 and to explore the mechanisms of phloretin induced apoptosis, this research is set to observed morphological alterations with phase contrast and confocal microscopy, performed MTT assay, and detected apoptotic rates, cell cycle progression, mitochondrial trans-membrane potential and intracellular calcium homeostasis. The results suggested that typical apoptotic morphological alterations occurred after phloretin treatment. Phloretin exerts a strong inhibitory on the proliferation of SMMC-7721 cell line, and induces its apoptosis in a dosage and duration dependent manner. Cell cycle was arrested at G1 phase. Mitochondrial trans-membrane potential dropped. Calcium homeostasis was disturbed. It is concluded that phloretin can induce apoptosis of SMMC-7721 via arresting cell cycle progression, reducing mitochondrial trans-membrane potential and disturbing intracellular calcium homeostasis. Key words: Apoptosis, phloretin, human hepatoma cells, SMMC-7721 cells.

WWOXinduces apoptosis and inhibits proliferation of human hepatoma cell line SMMC-7721

To investigate the effects of the WWOX gene on the human hepatic carcinoma cell line SMMC-7721. Full-length WWOX cDNA was amplified from normal human liver tissues. Full-length cDNA was subcloned into pEGFP-N1, a eukaryotic expression vector. After introduction of the WWOX gene into cancer cells using liposomes, the WWOX protein level in the cells was detected through Western blotting. Cell growth rates were assessed by methyl thiazolyl tetrazolium (MTT) and colony formation assays. Cell cycle progression and cell apoptosis were measured by flow cytometry. The phosphorylated protein kinase B (AKT) and activated fragments of caspase-9 and caspase-3 were examined by Western blotting analysis. WWOX significantly inhibited cell proliferation, as evaluated by the MTT and colony formation assays. Cells transfected with WWOX showed significantly higher apoptosis ratios when compared with cells transfected with a mock plasmid, and overexpression of WWOX delayed cell cycle progression from G1 to S phase, as measured by flow cytometry. An increase in apoptosis was also indicated by a remarkable activation of caspase-9 and caspase-3 and a dephosphorylation of AKT (Thr308 and Ser473) measured with Western blotting analysis. Overexpression of WWOX induces apoptosis and inhibits proliferation of the human hepatic carcinoma cell line SMMC-7721.