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1.  Schedule-dependent antitumor effects of 5-fluorouracil combined with sorafenib in hepatocellular carcinoma 
BMC Cancer  2013;13:363.
Recently, a phase II clinical trial in hepatocellular carcinoma (HCC) has suggested that the combination of sorafenib and 5-fluorouracil (5-FU) is feasible and side effects are manageable. However, preclinical experimental data explaining the interaction mechanism(s) are lacking. Our objective is to investigate the anticancer efficacy and mechanism of combined sorafenib and 5-FU therapy in vitro in HCC cell lines MHCC97H and SMMC-7721.
Drug effects on cell proliferation were evaluated by cell viability assays. Combined-effects analyses were conducted according to the median-effect principle. Cell cycle distribution was measured by flow cytometry. Expression levels of proteins related to the RAF/MEK/ERK and STAT3 pathways and to cell cycle progression (cyclin D1) were determined by western blot analysis.
Sorafenib and 5-FU alone or in combination showed significant efficacy in inhibiting cell proliferation in both cell lines tested. However, a schedule-dependent combined effect, associated with the order of compound treatments, was observed. Efficacy was synergistic with 5-FU pretreatment followed by sorafenib, but it was antagonistic with the reverse treatment order. Sorafenib pretreatment resulted in a significant increase in the half inhibitory concentration (IC50) of 5-FU in both cell lines. Sorafenib induced G1-phase arrest and significantly decreased the proportion of cells in S phase when administrated alone or followed by 5-FU. The RAF/MEK/ERK and STAT3 pathways were blocked and cyclin D1 expression was down regulated significantly in both cell lines by sorafenib; whereas, the kinase pathways were hardly affected by 5-FU, and cyclin D1 expression was up regulated.
Antitumor activity of sorafenib and 5-FU, alone or in combination, is seen in HCC cell lines. The nature of the combined effects, however, depends on the particular cell line and treatment order of the two compounds. Sorafenib appears to reduce sensitivity to 5-FU through down regulation of cyclin D1 expression by inhibiting RAF/MEK/ERK and STAT3 signaling, resulting in G1-phase arrest and reduction of the S-phase cell subpopulation when 5-FU is administrated after sorafenib, in which situation, combination treatment of the two agents results in antagonism; on the other hand, when sorafenib is administrated afterward, it can continue to work since it is not cell cycle specific, as a result, combination treatment of the two agents shows an additive-to-synergistic effect.
PMCID: PMC3734040  PMID: 23895220
Hepatocellular carcinoma; Sorafenib; 5-fluorouracil; Cell cycle arrest
2.  Synergistic Inhibitory Effect of Hyperbaric Oxygen Combined with Sorafenib on Hepatoma Cells 
PLoS ONE  2014;9(6):e100814.
Hypoxia is a common phenomenon in solid tumors, associated with chemotherapy and radiotherapy resistance, recurrence and metastasis. Hyperbaric oxygen (HBO) therapy can increase tissue oxygen pressure and content to prevent the resistance, recurrence and metastasis of cancer. Presently, Sorafenib is a first-line drug, targeted for hepatocellular carcinoma (HCC) but effective in only a small portion of patients and can induce hypoxia. The purpose of this study is to investigate the effect of HBO in combination with sorafenib on hepatoma cells.
Hepatoma cell lines (BEL-7402 and SK-Hep1) were treated with HBO at 2 atmosphere absolute pressure for 80 min per day or combined with sorafenib or cisplatin. At different time points, cells were tested for cell growth, colony formation, apoptosis, cell cycle and migration. Finally, miRNA from the hepatoma cells was detected by microRNA array and validated by qRT-PCR.
Although HBO, sorafenib or cisplatin alone could inhibit growth of hepatoma cells, HBO combined with sorafenib or cisplatin resulted in much greater synergistic growth inhibition (cell proliferation and colony formation) in hepatoma cells. Similarly, the synergistic effect of HBO and sorafenib on induction of apoptosis was also observed in hepatoma cells. HBO induced G1 arrest in SK-Hep1 not in BEL-7402 cells, but enhanced cell cycle arrest induced by sorafenib in BEL-7402 treated cells. However, HBO had no obvious effect on the migration of hepatoma cells, and microRNA array analysis showed that hepatoma cells with HBO treatment had significantly different microRNA expression profiles from those with blank control.
We show for the first time that HBO combined with sorafenib results in synergistic growth inhibition and apoptosis in hepatoma cells, suggesting a potential application of HBO combined with sorafenib in HCC patients. Additionally, we also show that HBO significantly altered microRNA expression in hepatoma cells.
PMCID: PMC4067386  PMID: 24956259
3.  Phosphorylated ERK is a potential predictor of sensitivity to sorafenib when treating hepatocellular carcinoma: evidence from an in vitro study 
BMC Medicine  2009;7:41.
Sorafenib is the first agent that has demonstrated an improved overall survival benefit in advanced hepatocellular carcinoma (HCC), setting a new standard for first-line treatment. However, no one has yet been able to predict sensitivity to sorafenib. Pre-treatment pERK level has been shown to be associated with favorable response to such therapy in a phase II clinical study, indicating that pERK may be a potential biomarker for treatment of HCC with sorafenib.
The effects of sorafenib and 5-fluorouracil (5-FU) on cell proliferation were evaluated by cell viability assays in four HCC cell lines (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6) with different metastatic potential and basal pERK expression levels. Expression levels of pERK were determined by immunocytochemical quantification together with western blot analysis, and pERK density values were also calculated. Correlation analyses were then carried out between the IC50 values of drugs and pERK density values. After basal ERK phosphorylation was down-regulated with U0126 in MHCC97-H cells, cellular responsiveness to sorafenib was assessed by cell viability assay.
Basal pERK levels increased stepwise in cell lines in accordance with their metastatic potential. Sorafenib inhibited ERK phosphorylation in a dose-dependent manner in all four cell lines at a concentration between 5 and 20 μM, but the degree of inhibition was significantly different according to their basal pERK expression level (P < 0.0001). In contrast, no significant change was observed after 5-FU treatment. Correlation analyses between the IC50 values and pERK densities revealed that the effects of sorafenib on cell proliferation were significantly correlated with basal pERK levels (Spearman r = -0.8671, P = 0.0003). Resistance to 5-FU was also significantly associated with basal pERK expression in these HCC cell lines (Spearman r = 0.7832, P = 0.0026). After the basal ERK phosphorylation level in MHCC97-H cells was reduced with U0126, they were significantly less sensitive to sorafenib-mediated growth inhibition, with an IC50 of 17.31 ± 1.62 μM versus 10.81 ± 1.24 μM (P = 0.0281).
In this in vitro study, pERK was confirmed to be a potential biomarker predictive of sensitivity to sorafenib in treating HCC. The RAF/MEK/ERK pathway may be involved in drug resistance to traditional chemotherapy in HCC.
PMCID: PMC2738687  PMID: 19698189
4.  YC-1 enhances the anti-tumor activity of sorafenib through inhibition of signal transducer and activator of transcription 3 (STAT3) in hepatocellular carcinoma 
Molecular Cancer  2014;13:7.
Traditional systemic chemotherapy does not provide survival benefits in patients with hepatocellular carcinoma (HCC). Molecular targeted therapy shows promise for HCC treatment, however, the duration of effectiveness for targeted therapies is finite and combination therapies offer the potential for improved effectiveness.
Sorafenib, a multikinase inhibitor, and YC-1, a soluble guanylyl cyclase (sGC) activator, were tested in HCC by proliferation assay, cell cycle analysis and western blot in vitro and orthotopic and ectopic HCC models in vivo.
In vitro, combination of sorafenib and YC-1 synergistically inhibited proliferation and colony formation of HepG2, BEL-7402 and HCCLM3 cells. The combination also induced S cell cycle arrest and apoptosis, as observed by activated PARP and caspase 8. Sorafenib and YC-1 respectively suppressed the expression of phosphorylated STAT3 (p-STAT3) (Y705) in a dose- and time-dependent manner. Combination of sorafenib and YC-1 significantly inhibited the expression of p-STAT3 (Y705) (S727), p-ERK1/2, cyclin D1 and survivin and SHP-1 activity compared with sorafenib or YC-1 used alone in all tested HCC cell lines. In vivo, sorafenib-YC-1 combination significantly suppressed the growth of HepG2 tumor xenografts with decreased cell proliferation and increased apoptosis observed by PCNA and PARP. Similar results were also confirmed in a HCCLM3 orthotopic model. There was a reduction in CD31-positive blood vessels and reduced VEGF expression, which suggested a combinational effect of sorafenib and YC-1 on angiogenesis. The reduced expression of p-STAT3, cyclin D1 and survivin was also observed with the combination of sorafenib and YC-1.
Our data show that sorafenib-YC-1 combination is a novel potent therapeutic agent that can target the STAT3 signaling pathway to inhibit HCC tumor growth.
PMCID: PMC3895679  PMID: 24418169
YC-1; Sorafenib; Hepatocellular carcinoma; STAT3
5.  Connexin-dependent gap junction enhancement is involved in the synergistic effect of sorafenib and all-trans retinoic acid on HCC growth inhibition 
Oncology Reports  2013;31(2):540-550.
Increasing gap junction activity in tumor cells provides a target by which to enhance antineoplastic therapies. Previously, several naturally occurring agents, including all-trans retinoic acid (ATRA) have been demonstrated to increase gap junctional intercellular communication (GJIC) in a number of types of cancer cells. In the present study, we investigated in vitro whether ATRA modulates the response of human hepatocellular carcinoma (HCC) cells to sorafenib, the only proven oral drug for advanced HCC, and the underlying mechanisms. HepG2 and SMMC-7721 cells were treated with sorafenib and/or ATRA, and cell proliferation and apoptosis were analyzed; the role of GJIC was also explored. We found that ATRA, at non-toxic concentrations, enhanced sorafenib-induced growth inhibition in both HCC cell lines, and this effect was abolished by two GJIC inhibitors, 18-α-GA and oleamide. Whereas lower concentrations of sorafenib (5 μM) or ATRA (0.1 or 10 μM) alone modestly induced GJIC activity, the combination of sorafenib plus ATRA resulted in a strong enhancement of GJIC. However, the action paradigm differed in the HepG2 and SMMC-7721 cells, with the dominant effect of GJIC dependent on the cell-specific connexin increase in protein amounts and relocalization. RT-PCR assay further revealed a transcriptional modification of the key structural connexin in the two cell lines. Thus, a connexin-dependent gap junction enhancement may play a central role in ATRA plus sorafenib synergy in inhibiting HCC cell growth. Since both agents are available for human use, the combination treatment represents a future profitable strategy for the treatment of advanced HCC.
PMCID: PMC3896525  PMID: 24317203
sorafenib; all-trans retinoic acid; growth inhibition; gap junction; hepatocellular carcinoma
6.  Salinomycin Inhibits Proliferation and Induces Apoptosis of Human Hepatocellular Carcinoma Cells In Vitro and In Vivo 
PLoS ONE  2012;7(12):e50638.
The anti-tumor antibiotic salinomycin (Sal) was recently identified as a selective inhibitor of breast cancer stem cells; however, the effect of Sal on hepatocellular carcinoma (HCC) is not clear. This study aimed to determine the anti-tumor efficacy and mechanism of Sal on HCC. HCC cell lines (HepG2, SMMC-7721, and BEL-7402) were treated with Sal. Cell doubling time was determinated by drawing growth curve, cell viability was evaluated using the Cell Counting Kit 8. The fraction of CD133+ cell subpopulations was assessed by flow cytometry. We found that Sal inhibits proliferation and decreases PCNA levels as well as the proportion of HCC CD133+cell subpopulations in HCC cells. Cell cycle was analyzed using flow cytometry and showed that Sal caused cell cycle arrest of the various HCC cell lines in different phases. Cell apoptosis was evaluated using flow cytometry and Hoechst 33342 staining. Sal induced apoptosis as characterized by an increase in the Bax/Bcl-2 ratio. Several signaling pathways were selected for further mechanistic analyses using real time-PCR and Western blot assays. Compared to control, β-catenin expression is significantly down-regulated upon Sal addition. The Ca2+ concentration in HCC cells was examined by flow cytometry and higher Ca2+ concentrations were observed in Sal treatment groups. The anti-tumor effect of Sal was further verified in vivo using the hepatoma orthotopic tumor model and the data obtained showed that the size of liver tumors in Sal-treated groups decreased compared to controls. Immunohistochemistry and TUNEL staining also demonstrated that Sal inhibits proliferation and induces apoptosis in vivo. Finally, the role of Sal on in vivo Wnt/β-catenin signaling was evaluated by Western blot and immunohistochemistry. This study demonstrates Sal inhibits proliferation and induces apoptosis of HCC cells in vitro and in vivo and one potential mechanism is inhibition of Wnt/β-catenin signaling via increased intracellular Ca2+ levels.
PMCID: PMC3527475  PMID: 23284640
7.  Aberrant methylation and downregulation of sall3 in human hepatocellular carcinoma 
AIM: To investigated whether sall3 transcription was regulated by promoter CpG island hypermethylation in hepatocellular carcinoma (HCC).
METHODS: The cell lines Huh7, HepG2, SK-HEP1, SMMC7721, Bel7402, QGY7703 and a cohort of 38 HCC tissue specimens and corresponding nontumorous tissues were subjected to analysis for sall3 promoter CpG island methylation and mRNA transcription. sall3 promoter CpG island methylation levels were determined using the MassARRAY platform and mRNA transcription levels of the gene were detected by quantitative real-time polymerase chain reaction.
RESULTS: The levels of sall3 mRNA were decreased by more than twofold in 33 of 38 tumor tissues compared to adjacent noncancerous tissues. Among these 33 tumor tissues with lower levels of sall3 mRNA, 24 showed higher levels of methylation. Based on these results, we hypothesized that the decrease in sall3 mRNA transcription level was likely due to promoter CpG island hypermethylation. Changes in sall3 mRNA transcription and promoter CpG island methylation were determined in the above six cell lines after treatment with 0, 0.1, 0.5 and 2.5 μmol 5-aza-2-deoxycytidine, a demethylating agent. Promoter CpG island methylation levels decreased in a dose-dependent manner in all six cell lines, while the mRNA transcription level increased dose-dependently in Huh7, HepG2, SK-HEP1 and SMMC7721 cells and irregularly in Bel7402 and QGY7703 cells.
CONCLUSION: These results indicated that promoter CpG island hypermethylation contributes to the downregulation of sall3 mRNA transcription in HCC.
PMCID: PMC3370011  PMID: 22690083
Hepatocellular carcinoma; sall3; Aberrant methylation; Down regulation mRNA transcription
8.  Smac Mimetic SM-164 Potentiates APO2L/TRAIL- and Doxorubicin-Mediated Anticancer Activity in Human Hepatocellular Carcinoma Cells 
PLoS ONE  2012;7(12):e51461.
The members of inhibitor of apoptosis proteins (IAPs) family are key negative regulators of apoptosis. Overexpression of IAPs are found in hepatocellular carcinoma (HCC), and can contribute to chemotherapy resistance and recurrence of HCC. Small-molecule Second mitochondria-derived activator of caspases (Smac) mimetics have recently emerged as novel anticancer drugs through targeting IAPs. The specific aims of this study were to 1) examine the anticancer activity of Smac mimetics as a single agent and in combination with chemotherapy in HCC cells, and 2) investigate the mechanism of anticancer action of Smac mimetics.
Four HCC cell lines, including SMMC-7721, BEL-7402, HepG2 and Hep3B, and 12 primary HCC cells were used in this study. Smac mimetic SM-164 was used to treat HCC cells. Cell viability, cell death induction and clonal formation assays were used to evaluate the anticancer activity. Western blotting analysis and a pancaspase inhibitor were used to investigate the mechanisms.
Although SM-164 induced complete cIAP-1 degradation, it displayed weak inhibitory effects on the viability of HCC cells. Nevertheless, SM-164 considerably potentiated Apo2 ligand or TNF-related apoptosis-inducing ligand (APO2L/TRAIL)- and Doxorubicin-mediated anticancer activity in HCC cells. Mechanistic studies demonstrated that SM-164 in combination with chemotherapeutic agents resulted in enhanced activation of caspases-9, -3 and cleavage of poly ADP-ribose polymerase (PARP), and also led to decreased AKT activation.
Smac mimetics can enhance chemotherapeutic-mediated anticancer activity by enhancing apoptosis signaling and suppressing survival signaling in HCC cells. This study suggests Smac mimetics are potential therapeutic agents for HCC.
PMCID: PMC3519882  PMID: 23240027
9.  8-bromo-7-methoxychrysin-induced apoptosis of hepatocellular carcinoma cells involves ROS and JNK 
AIM: To investigate whether the apoptotic activities of 8-bromo-7-methoxychrysin (BrMC) involve reactive oxygen species (ROS) generation and c-Jun N-terminal kinase (JNK) activation in human hepatocellular carcinoma cells (HCC).
METHODS: HepG2, Bel-7402 and L-02 cell lines were cultured in vitro and the apoptotic effects of BrMC were evaluated by flow cytometry (FCM) after propidium iodide (PI) staining, caspase-3 activity using enzyme-linked immunosorbent assay (ELISA), and DNA agarose gel electrophoresis. ROS production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCHF-DA) probe labeling. The phosphorylation level of JNK and c-Jun protein was analyzed by Western blotting.
RESULTS: FCM after PI staining showed a dose-dependent increase in the percentage of the sub-G1 cell population (P < 0.05), reaching 39.0% ± 2.8% of HepG2 cells after 48 h of treatment with BrMC at 10 μmol/L. The potency of BrMC to HepG2 and Bel-7402 (32.1% ± 2.6%) cells was found to be more effective than the lead compound, chrysin (16.2% ± 1.6% for HepG2 cells and 11.0% ± 1.3% for Bel-7402 cell) at 40 μmol/L and similar to 5-flurouracil (33.0% ± 2.1% for HepG2 cells and 29.3% ± 2.3% for Bel-7402 cells) at 10 μmol/L. BrMC had little effect on human embryo liver L-02 cells, with the percentage of sub-G1 cell population 5.4% ± 1.8%. Treatment of HepG2 cells with BrMC for 48 h also increased the levels of active caspase-3, in a concentration-dependent manner. z-DEVD-fmk, a caspase-3-specific inhibitor, prevented the activation of caspase-3. Treatment with BrMC at 10 μmol/L for 48 h resulted in the formation of a DNA ladder. Treatment of cells with BrMC (10 μmol/L) increased mean fluorescence intensity of DCHF-DA in HepG2 cells from 7.2 ± 1.12 at 0 h to 79.8 ± 3.9 at 3 h and 89.7 ± 4.7 at 6 h. BrMC did not affect ROS generation in L-02 cells. BrMC treatment failed to induce cell death and caspase-3 activation in HepG2 cells pretreated with N-acetylcysteine (10 mmol/L). In addition, in HepG2 cells treated with BrMC (2.5, 5.0, 10.0 μmol/L) for 12 h, JNK activation was observed. Peak JNK activation occurred at 12 h post-treatment and this activation persisted for up to 24 h. The expression of phosphorylated JNK and c-Jun protein after 12 h with BrMC-treated cells was inhibited by N-acetylcysteine and SP600125 pre-treatment, but GW9662 had no effect. SP600125 substantially reduced BrMC-induced cell death and caspase-3 activation of HepG2 cells. N-acetylcysteine and GW9662 also attenuated induction of cell death and caspase-3 activation in HepG2 cells treated with BrMC.
CONCLUSION: BrMC induces apoptosis of HCC cells by ROS generation and sustained JNK activation.
PMCID: PMC2904884  PMID: 20632440
Hepatocellular carcinoma; 8-bromo-7-methoxychysin; Chrysin; Reactive oxygen species; Jun N-terminal kinase
10.  Maintenance of Sorafenib following combined therapy of three-dimensional conformal radiation therapy/intensity-modulated radiation therapy and transcatheter arterial chemoembolization in patients with locally advanced hepatocellular carcinoma: a phase I/II study 
Three-dimensional conformal radiation therapy (3DCRT)/intensity-modulated radiation therapy (IMRT) combined with or without transcatheter arterial chemoembolization (TACE) for locally advanced hepatocellular carcinoma (HCC) has shown favorable outcomes in local control and survival of locally advanced HCC. However, intra-hepatic spreading and metastasis are still the predominant treatment failure patterns. Sorafenib is a multikinase inhibitor with effects against tumor proliferation and angiogenesis. Maintenance Sorafenib would probably prevent or delay the intrahepatic and extrahepatic spread of HCC after radiotherapy, which provides the rationale for the combination of these treatment modalities.
Methods and design
Patients with solitary lesion (bigger than 5 cm in diameter) histologically or cytologically confirmed HCC receive TACE (1-3 cycles) plus 3DCRT/IMRT 4-6 weeks later. Maintenance Sorafenib will be administered only for the patients with non-progression disease 4 to 6 weeks after the completion of radiotherapy. The dose will be 400 mg, p.o., twice a day. Sorafenib will be continuously given for 12 months unless intolerable toxicities and/or tumor progression. If no more than 3 patients discontinue Sorafenib treatment who experience dose-limiting toxicity after necessary dose modification and delay and/or radiation-induced liver disease in the first 15 enrolled patients, the study will recruit second fifteen patients for further evaluating safety and efficacy of treatment. Hypothesis of the current study is that Sorafenib as a maintenance therapy after combined therapy of 3DCRT/IMRT and TACE is safe and superior to radiotherapy combined with TACE alone in terms of time to progression (TTP), progression-free survival (PFS) and overall survival (OS) in comparison to historical data.
A recent meta-analysis showed TACE in combination with radiotherapy, improved the survival and the tumor response of patients, and was thus more therapeutically beneficial. In this study, local therapy for HCC is the combination of TACE and radiotherapy. Radiation exposure as a kind of stress might induce the compensatory activations of multiple intracellular signaling pathway mediators, such as PI3K, MAPK, JNK and NF-kB. Vascular endothelial growth factor (VEGF) was identified as one factor that was increased in a time- and dose-dependent manner after sublethal irradiation of HCC cells in vitro, translating to enhanced intratumor angiogenesis in vivo. Therefore, Sorafenib-mediated blockade of the Raf/MAPK and VEGFR pathways might enhance the efficacy of radiation, when Sorafenib is followed sequentially as a maintenance modality. ( number, NCT00999843.)
PMCID: PMC2829587  PMID: 20149262
11.  Numb Promotes Cell Proliferation and Correlates with Poor Prognosis in Hepatocellular Carcinoma 
PLoS ONE  2014;9(4):e95849.
Numb is an evolutionary conserved protein that plays critical roles in cell fate determination, cell adhesion, cell migration and a number of signaling pathways, but evidence for a substantial involvement of Numb in HCC has remained unclear. The present study was aimed to investigate the clinical and prognostic significance of Numb and its role in hepatocellular carcinoma (HCC).
The expression of Numb was detected in 107 cases of clinical paraffin-embedded hepatocellular carcinoma tissues,5 matched paris of fresh tissues and six hepatocellular cell lines by immunohistochemistry with clinicopathological analyses,RT-PCR or Western blot. Moreover, loss of function and gain of function assays were performed to evaluate the effect of Numb on cell proliferation in vitro.
We found that Numb was obviously up-regulated in HCC tissues and cell lines (p<0.05). The Numb up-regulation correlated significantly with poor prognosis, and Numb status was identified as an independent prognostic factor. Over-expression of Numb increased proliferation in SMMC-7721 and BEL-7402 cells, while knock-down of Numb showed the opposite effect. Our study indicates that Numb up-regulation significantly correlates with cell proliferation and poor prognosis in hepatocellular carcinoma patients. It may be a useful biomarker for therapeutic strategy in hepatocellular carcinoma treatment.
PMCID: PMC4000222  PMID: 24770339
12.  Down-regulation of extracellular signal-regulated kinase 1/2 activity in P-glycoprotein-mediated multidrug resistant hepatocellular carcinoma cells 
AIM: To study the expression and phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2 in multidrug resistant (MDR) hepatocellular carcinoma (HCC) cells.
METHODS: MDR HCC cell lines, HepG2/adriamycin (ADM) and SMMC7721/ADM, were developed by exposing parental cells to stepwise increasing concentrations of ADM. MTT assay was used to determine drug sensitivity. Flow cytometry was employed to analyze cell cycle distribution and measure cell P-glycoprotein (P-gp) and multidrug resistant protein 1 (MRP1) expression levels. ERK1 and ERK2 mRNA expression levels were measured by quantitative real-time PCR (QRT-PCR). Expression and phosphorylation of ERK1 and ERK2 were analyzed by Western blot.
RESULTS: MTT assay showed that HepG2/ADM and SMMC7721/ADM were resistant not only to ADM, but also to multiple anticancer drugs. The P-gp expression was over 10-fold higher in HepG2/ADM cells than in HepG2 cells (8.92% ± 0.22% vs 0.88% ± 0.05%, P < 0.001) and over 4-fold higher in SMMC7721/ADM cells than in SMMC7721 cells (7.37% ± 0.26% vs 1.74% ± 0.25%, P < 0.001). However, the MRP1 expression was not significantly higher in HepG2/ADM and SMMC7721/ADM cells than in parental cells. In addition, the percentage of MDR HepG2/ADM and SMMC7721/ADM cells was significantly decreased in the G0/G1 phase and increased in the the S phase or G2/M phase. QRT-PCR analysis demonstrated that the ERK1 and ERK2 mRNA expression increased apparently in HepG2/ADM cells and decreased significantly in SMMC7721/ADM cells. Compared with the expression of parental cells, ERK1 and ERK2 protein expressions were markedly decreased in SMMC7721/ADM cells. However, ERK2 protein expression was markedly increased while ERK1 protein expression had no significant change in HepG2/ADM cells. Phosphorylation of ERK1 and ERK2 was markedly decreased in both HepG2/ADM and SMMC7721/ADM MDR cells.
CONCLUSION: ERK1 and ERK2 activities are down-regulated in P-gp-mediated MDR HCC cells. ERK1 or ERK2 might be a potential drug target for circumventing MDR HCC cells.
PMCID: PMC2665138  PMID: 19322916
Multidrug resistance; Extracellular signal-regulated MAP kinases; Hepatocellular carcinoma; P-glycoprotein; Multidrug resistance-associated protein
13.  Mechanism of enhancement of radiation-induced cytotoxicity by sorafenib in colorectal cancer 
Journal of Radiation Research  2012;54(1):52-60.
Sorafenib, an orally available multikinase inhibitor, combined with radiation has shown potential as an anticancer treatment in an in vitro and in vivo colon cancer model. In this study, we investigated the mechanism of enhancement of radiation-induced cytotoxicity by sorafenib in colorectal cancer. The effects of sorafenib on radiation-induced cytotoxicity of DLD-1 and HT-29 were evaluated via clonogenic assay. The impact of sorafenib on radiation-induced cell cycle kinetics and on apoptosis was analyzed using flow cytometry. Cyclin B1 was examined by western blot. As a measure of DNA damage after treatment, γ-H2AX foci and nuclear fragmentation were determined as a function of time after irradiation plus sorafenib combination. Tumor growth delay was used to evaluate the effects of sorafenib on in vivo radiation-induced cytotoxicity. Exposure of each cell line to sorafenib combined with irradiation resulted in an increased radiation-induced cytotoxicity with dose enhancement factors at a surviving fraction of 0.37 ranging from 1.13 to 1.76. Sorafenib strengthened radiation-induced accumulation of tumor cells in the G2-M phase with attenuated expression of cyclin B1, but had no effect on radiation-induced apoptosis. Exposure to sorafenib and radiation resulted in a greater number of remaining γ-H2AX foci and fragmented nuclei than radiation alone. In vivo tumor xenograft study confirmed that administration of sorafenib results in significant tumor growth inhibition when combined with radiation. These results indicate that sorafenib enhances radiation-induced cytotoxicity in colorectal cancer and suggest that the mechanism is associated with delaying repair of radiation-induced DNA damage and down-regulation of cyclin B1.
PMCID: PMC3534276  PMID: 22923745
radiation; sorafenib; colorectal cancer; DNA damage; cell cycle
14.  Comparison of the multi-drug resistant human hepatocellular carcinoma cell line Bel-7402/ADM model established by three methods 
To compare the biological characteristics of three types of human hepatocellular carcinoma multi-drug resistant cell sub-lines Bel-7402/ADM models established by three methods.
Established human hepatocellular carcinoma adriamycin (ADM) multi-drug resistant cell sub-lines models Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS by three methods of in vitro concentration gradient increased induction, nude mice liver-implanted induction and subcutaneous-implanted induction respectively. Phase contrast microscopy was used to observe the cells and the MTT (methyl thiazolyl tetrazolium) method was used to detect drug resistance of the three different sub-lines of cells.
The three groups of drug resistant cells, Bel-7402/ADMV, Bel-7402/ADML and Bel-7402/ADMS generated cross-resistance to ADM and CDDP (cis-Diaminedichloroplatinum), but showed a significant difference in resistance to Bel-7402 IC50 value (P < 0.01). The doubling times were significantly extended compared to the parent cell line (39 h) and were 65 h (Bel-7402/ADMV), 46 h (Bel-7402/ADML), and 45 h (Bel-7402/ADMS). The excretion rates of ADM were significantly increased compared with the parent cell (34.14%) line and were 81.06% (Bel-7402/ADMV), 66.56% (Bel-7402/ADML) and 61.56% (Bel-7402/ADMS). Expression of P-gp and MRP in the three groups of resistant cells was significantly enhanced (P < 0.01). There was no significant variation in the expression of GSH/GST (P > 0.05).
Stable resistance was involved in the resistant cell line model established by the above three methods. Liver implantation was a good simulation of human hepatocellular and proved to be an ideal model with characteristics similar to human hepatocellular biology and the pharmacokinetics of anticancer drugs.
PMCID: PMC2933613  PMID: 20727186
15.  The novel mTOR inhibitor CCI-779 (temsirolimus) induces antiproliferative effects through inhibition of mTOR in Bel-7402 liver cancer cells 
Liver cancer is one of the most frequent cancers in the world. Targeted therapy of cancer with specific inhibitors is developing and has shown promising antitumor efficacy. CCI-779 (temsirolimus), a specific inhibitor of mTOR (mammalian target of rapamycin), can block the mTOR signaling pathway. Here, we systematically examined the expression of mTOR and its downstream targets in liver cancer cells and normal liver cells, then investigated inhibitory effects of CCI-779 on mTOR signaling pathway and its role in regulating liver cancer cell growth.
The expression of mTOR and its downstream targets in Bel-7402 liver cancer cells and HL-7702 normal liver cells were examined by western blot. The mTOR specific inhibitor (CCI-779) was used to treat Bel-7402 cells to identify its effects on Bel-7402 cell growth and activity of mTOR signaling pathway in vitro. Cell viability tests were performed after the treatment of CCI-779. Western blot was applied to assess the changes of mTOR pathway and flow cytometry was used to analyze cell cycle of Bel-7402 cells after the treatment of CCI-779.
mTOR, p70S6K, S6, and 4EBP1 were overexpressed in Bel-7402 cells compared with HL-7702 cells. Bel-7402 cells were sensitive to CCI-779. The survival rate of the cells treated with CCI-779 over 0.312 μM was significantly different compared with that of control (P < 0.05). CCI-779 inhibited the phosphorylation of mTOR (Ser2448), p70S6K (Thr389), S6 (Ser240/244), and 4EBP1 (Thr37/46) in different grades and the expressions of p70S6K, S6, and 4EBP1. As a result, CCI-779 induced a dose-dependent decrease in cell proliferation, G1/S arrest and damage of cell shape.
Taken together, these data showed that CCI-779 can inhibit mTOR signaling and proliferation in Bel-7402 liver cancer cells in vitro. It offers a therapeutic intervention through inhibition of mTOR as a potential strategy for liver cancer.
PMCID: PMC3632488  PMID: 23537100
CCI-779 (temsirolimus); mTOR signaling; Cell growth; Liver cancer cell
16.  Induction of an EMT-like transformation and MET in vitro 
The epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) play pivotal roles in metastasis of epithelial cancers. The distinction between them has shed new light on the molecular mechanisms of tumor metastasis. Recently, tumor microenvironment (TM) has been identified as one of the most potent inducers of EMT and MET. TM is characterized by its complexity and flexibility. The purpose of this study was to ascertain the exact effect of each distinct TM component on the evolution hepatocellular carcinoma (HCC) metastasis.
Two different cell culture models were used. The HCC cell line Bel-7402 was co-cultured with the normal liver cell line HL-7702 or with the retinal vascular endothelial cell line RF/6A in double-layer six-well plates, imitating the direct interaction between tumor-host cells and tumor cells. Bel-7402 was also cultured in the conditioned medium (CM) of the human lung fibroblast cell line MRC-5, HL-7702 or RF/6A, imitating an indirect interaction. Integrin β1, β3, β4, β7, laminin β3, E-cadherin and Snail levels were measured by quantitative RT-PCR in tumor sepecimens from 42 resected HCC.
We found that Bel-7402 cells co-cultured with HL-7702 or RF/6A cells were induced to undergo MET. The expression of E-cadherin, α-catenin and β-catenin was up-regulated, accompanied with a strengthened E-cadherin/catenin complex on the membrane of co-cultured Bel-7402 cells. Consequently, the invasion and migration ability of cells was declined. Conversely, Bel-7402 cells cultured in conditioned medium from MRC-5 cells underwent an EMT-like transformation as the cells became elongated with increased invasion and migration ability. Furthermore, we demonstrated that HL-7702 cells could generally inhibit the tumorigenicity and viability of Bel-7402 cells. We also found that integrin β1 expression was negatively associated with capsular formation, and that integrin β4 expression was negatively associated with CK19 expression.
Our findings highlight the strong influences exerted by TM on tumor progression through EMT and MET by impacting the expression of adhesion molecules, including the E-cadherin/catenin complex, laminins and integrins.
PMCID: PMC3716679  PMID: 23829659
Tumor microenvironment; EMT; MET; Co-culture; Conditioned medium culture
17.  Hydroxysteroid Sulfotransferase SULT2B1b Promotes Hepatocellular Carcinoma Cells Proliferation In Vitro and In Vivo 
PLoS ONE  2013;8(4):e60853.
Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) is highly selective for the addition of sulfate groups to 3β-hydroxysteroids. Although previous reports have suggested that SULT2B1b is correlated with cell proliferation of hepatocytes, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. In the present study, we found that SULT2B1 was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Besides, SULT2B1b overexpression promoted the growth of the mouse hepatocarcinoma cell line Hepa1-6, while Lentivirus-mediated SULT2B1b interference inhibited growth as assessed by the CCK-8 assay. Likewise, inhibition of SULT2B1b expression induced cell-cycle arrest and apoptosis in Hepa1-6 cells by upregulating the expression of FAS, downregulating the expression of cyclinB1, BCL2 and MYC in vitro and in vivo at both the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in nude mouse xenografts. Moreover, proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Knock-down of SULT2B1b inhibited cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppressed xenograft growth in vivo. In conclusion, SULT2B1b expression promotes proliferation of hepatocellular carcinoma cells in vitro and in vivo, which may contribute to the progression of HCC.
PMCID: PMC3623875  PMID: 23593328
18.  Apoptosis selectively induced in BEL-7402 cells by folic acid-modified magnetic nanoparticles combined with 100 Hz magnetic field 
To explore the effect of folic acid-modified magnetic nanoparticles (FA-MNPs) combined with a 100 Hz extremely low-frequency electromagnetic field (ELF-EMF) on the apoptosis of liver cancer BEL-7402 cells.
Materials and methods
MNPs (20 nm) were prepared by coprecipitation, and then folic acid was coated onto MNPs to prepare FA-MNPs. BEL-7402 cells and HL7702 cells were selected as liver cancer cells and normal liver cells, respectively. The ELF-EMF was generated from a solenoid coil. Cellular uptake of NPs was determined by inductively coupled plasma atomic emission spectroscopy. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to evaluate cell inhibition. Apoptosis was analyzed by flow cytometry. Statistical analyses were performed using two-way analysis of variance.
FA-MNPs combined with a 100 Hz magnetic field significantly inhibited cell proliferation and induced higher apoptosis compared to either the ELF-EMF alone or FA-MNPs alone. FA-MNPs showed a better apoptosis effect and higher iron uptake in BEL-7402 cells compared to in HL7702 cells. On the basis of the ELF-EMF, higher doses of FA-MNPs brought higher apoptosis and higher iron uptake in either BEL-7402 cells or HL7702 cells.
These results suggest that FA-MNPs may induce apoptosis in a cellular iron uptake-dependent manner when combined with an ELF-EMF in BEL-7402 cells.
PMCID: PMC4003272  PMID: 24790442
extremely low-frequency magnetic field; magnetic nanoparticle; apoptosis; liver cancer; folic acid
19.  Insufficient radiofrequency ablation promotes epithelial-mesenchymal transition of hepatocellular carcinoma cells through Akt and ERK signaling pathways 
Residual tumor progression after insufficient radiofrequency ablation (RFA) has been recently reported. However, whether epithelial-mesenchymal transition (EMT), which is a key process that drives cancer metastasis, is involved in the tumor progression after insufficient RFA is not well understood.
Human hepatocellular carcinoma (HCC) cell lines SMMC7721 and Huh7 were used. Insufficient RFA was simulated using a water bath (47°C 5 min, 10 min, 15 min, 20 min and 25 min gradually). MTT assay was used to evaluate the proliferation of HCC cells in vitro. Migration and invasion of HCC cells were determined by transwell assay. The molecular changes in HCC cells after insufficient RFA were evaluated by western blot. LY294002 and PD98059 were used to treat HCC cells. An ectopic nude mice model and a tail vein metastatic assay were used to evaluate the growth and metastatic potential of SMMC7721 cells in vivo after insufficient RFA.
SMMC7721 and Huh7 cells after insufficient RFA (named as SMMC7721-H and Huh7-H respectively) exhibited enhanced proliferation, migration and invasion (6.4% and 23.6%, 33.2% and 66.1%, and 44.1% and 57.4% increase respectively) in vitro. Molecular changes of EMT were observed in SMMC7721-H and Huh7-H cells. LY294002 and PD98059 inhibited the EMT of SMMC7721-H and Huh7-H cells. SMMC7721-H cells also exhibited larger tumor size (1440.8 ± 250.3 mm3 versus 1048.56 ± 227.6 mm3) and more lung metastasis (97.4% increase) than SMMC7721 cells in vivo. Higher expression of PCNA, N-cadherin and MMP-2 and MMP-9, was also observed in SMMC7721-H tumors.
Insufficient RFA could directly promote the invasiveness and metastasis of HCC cells. Insufficient RFA may promote the EMT of HCC cells through Akt and ERK signaling pathways.
PMCID: PMC3842745  PMID: 24168056
Insufficient radiofrequency ablation; Epithelial-mesenchymal transition; Hepatocellular carcinoma; Metastasis
20.  Autophagy induced by baicalin involves downregulation of CD147 in SMMC-7721 cells in vitro 
Oncology Reports  2011;27(4):1128-1134.
Baicalin has been demonstrated to exert anticancer effects mainly through induction of tumor cell apoptosis and cell cycle arrest. However, the precise mechanisms underlying its anticancer role remain to be elucidated. In the present study, we investigated whether autophagy was involved in the anticancer activity of baicalin in the human hepatocellular carcinoma (HCC) cell line SMMC-7721 and the possible molecular mechanisms. Our data showed that the viability of SMMC-7721 cells was significantly inhibited by baicalin in a dose- and time-dependent manner. Alongside apoptosis, autophagy was also induced by baicalin dose- and time-dependently with the involvement of the autophagy-associated protein Beclin 1. Moreover, we demonstrated that cell death induced by baicalin was significantly inhibited by the apoptosis inhibitor z-DEVD-fmk or the autophagy inhibitor 3-MA, respectively. In addition, we found that CD147, a key molecule related both to apoptosis and autophagy, was markedly downregulated at the protein level in SMMC-7721 cells treated with baicalin. Collectively, this is the first study to suggest that baicalin induces autophagic cell death in SMMC-7721 cells, which involves the downregulation of CD147. Our study reveals a new mechanism for the anticancer effects of baicalin and puts forward a potential crucial role of CD147 in baicalin-induced cancer cell death.
PMCID: PMC3583557  PMID: 22200845
baicalin; hepatocellular carcinoma cell; autophagy; apoptosis; CD147
21.  Recombinant vascular basement-membrane-derived multifunctional peptide inhibits angiogenesis and growth of hepatocellular carcinoma 
AIM: To investigate the anti-angiogenic and anti-tumor activities of recombinant vascular basement membrane-derived multifunctional peptide (rVBMDMP) in hepatocellular carcinoma (HCC).
METHODS: HepG2, Bel-7402, Hep-3B, HUVE-12 and L-02 cell lines were cultured in vitro and the inhibitory effect of rVBMDMP on proliferation of cells was detected by MTT assay. The in vivo antitumor efficacy of rVBMDMP on HCC was assessed by HepG2 xenografts in nude mice. Distribution of rVBMDMP, mechanism by which the growth of HepG2 xenografts is inhibited, and microvessel area were observed by proliferating cell nuclear antigen (PCNA) and CD31 immunohistochemistry.
RESULTS: MTT assay showed that rVBMDMP markedly inhibited the proliferation of human HCC (HepG2, Bel-7402, Hep-3B) cells and human umbilical vein endothelial (HUVE-12) cells in a dose-dependent manner, with little effect on the growth of L-02 cells. When the IC50 was 4.68, 7.65, 8.96, 11.65 and 64.82 μmol/L, respectively, the potency of rVBMDMP to HepG2 cells was similar to 5-fluorouracil (5-FU) with an IC50 of 4.59 μmol/L. The selective index of cytotoxicity to HepG2 cells of rVBMDMP was 13.8 (64.82/4.68), which was higher than that of 5-FU [SI was 1.9 (8.94/4.59)]. The VEGF-targeted recombinant humanized monoclonal antibody bevacizumab (100 mg/L) did not affect the proliferation of HepG2, Bel-7402, Hep-3B and L-02 cells, but the growth inhibitory rate of bevacizumab (100 mg/L) to HUVE-12 cells was 87.6% ± 8.2%. Alternis diebus intraperitoneal injection of rVBMDMP suppressed the growth of HepG2 xenografts in a dose-dependent manner. rVBMDMP (1, 3, 10 mg/kg) decreased the tumor weight by 12.6%, 55.9% and 79.7%, respectively, compared with the vehicle control. Immunohistochemical staining of rVBMDMP showed that the positive area rates (2.2% ± 0.73%, 4.5% ± 1.3% and 11.5% ± 3.8%) in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly higher than that (0.13% ± 0.04%) in the control group (P < 0.01). The positive area rates (19.0% ± 5.7%, 12.2% ± 3.5% and 5.2% ± 1.6% ) of PCNA in rVBMDMP treated group (1, 3, 10 mg/kg) were significantly lower than that (29.5% ± 9.4%) in the control group (P < 0.05). rVBMDMP at doses of 1, 3 and 10 mg/kg significantly reduced the tumor microvessel area levels (0.26% ± 0.07%, 0.12% ± 0.03% and 0.05% ± 0.01% vs 0.45% ± 0.15%) in HepG2 xenografts (P < 0.01), as assessed by CD31 staining.
CONCLUSION: rVBMDMP has effective and unique anti-tumor properties, and is a promising candidate for the development of anti-tumor drugs.
PMCID: PMC2668780  PMID: 19360918
Hepatocellular carcinoma; Recombinant vascular basement membrane-derived multifunctional peptide; Proliferating cell nuclear antigen; CD31; Therapeutic action
22.  Tumor suppressor function of ezrin-radixin-moesin-binding phosphoprotein-50 through β-catenin/E-cadherin pathway in human hepatocellular cancer 
AIM: To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50 (EBP50) in hepatocellular carcinoma (HCC).
METHODS: Three human HCC cell lines, i.e., SM-MC7721, HepG2 and Hep3B, were used. We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50. Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells. In vitro cell proliferation was assessed with a Cell Counting Kit-8 (CCK-8) assay. Cell cycle distribution was assessed with flow cytometry. Invasion and migration ability of before and after the transfection were determined with a transwell assay. Cell apoptosis was demonstrated with Annexin V-FITC. The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.
RESULTS: The transfection efficiency was confirmed with Western blotting (1.36 ± 0.07 vs 0.81 ± 0.09, P < 0.01). The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells (P < 0.01). Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50 (61.3% ± 3.1% vs 54.0% ± 2.4%, P < 0.05). The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells (5.8 ± 0.8 vs 21.6 ± 1.3, P < 0.01). Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells (14.8% ± 2.7% vs 3.4% ± 1.3%, P < 0.05). The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells (0.28 ± 0.07 vs 0.56 ± 0.12, P < 0.05; 0.55 ± 0.08 vs 0.39 ± 0.07, P < 0.05). In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells (28.9 ± 7.2 vs 70.1 ± 7.2, P < 0.01).
CONCLUSION: The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin, E-cadherin. EBP50 may serve asa potential therapeutic target in HCC.
PMCID: PMC3587489  PMID: 23483729
Hepatocellular carcinoma; Ezrin-radixin-moesin-binding phosphoprotein-50; Growth; Migration; Invasion
23.  BORIS, Brother of the Regulator of Imprinted Sites, Is Aberrantly Expressed in Hepatocellular Carcinoma 
Background: The brother of the regulator of imprinted sites (BORIS) is a novel member of the cancer testis antigen gene family, which are normally expressed only in spermatocytes, but abnormally activated in different malignancies. Aim: The aim of this study was to explore the expression of BORIS in hepatocellular carcinoma (HCC) and its correlation with the clinicopathologic features and prognosis of HCC. Methods: We investigated BORIS expression in HCC cell lines and 105 primary HCC clinical surgical specimens using real-time polymerase chain reaction and Western blot analysis. We further examined the correlation of BORIS with a liver stem cell marker (CD90) in HCC tissues by histochemical double staining. The correlation of BORIS with clinicopathologic features and prognosis of HCC was analyzed using patient data. Results: The expression of BORIS was found in SMMC-7721, BEL-7402, and Huh-7, but not in hep-G2 cells. The expression rate of BORIS was significantly higher in the HCC tissues than in the adjacent noncancerous tissues (p=0.000). BORIS expression was correlated with the tumor size (p=0.000), CD90 expression (p=0.000), and satellite nodule (p=0.000). Kaplan–Meier survival curves showed that patients with positive expression of BORIS had lower overall survival rate (p=0.003). Conclusions: Our data indicate that BORIS may be an auxiliary diagnosis index and a novel favorable prognostic indicator of HCC.
PMCID: PMC3552170  PMID: 23237599
24.  Downregulation of CD147 expression alters cytoskeleton architecture and inhibits gelatinase production and SAPK pathway in human hepatocellular carcinoma cells 
CD147 plays a critical role in the invasive and metastatic activity of hepatocellular carcinoma (HCC) cells by stimulating the surrounding fibroblasts to express matrix metalloproteinases (MMPs). Tumor cells adhesion to extracellular matrix (ECM) proteins is the first step to the tumor metastasis. MMPs degrade the ECM to promote tumor metastasis. The aim of this study is to investigate the effects of small interfering RNA (siRNA) against CD147 (si-CD147) on hepatocellular carcinoma cells' (SMMC-7721) architecture and functions.
Flow cytometry and western blot assays were employed to detect the transfection efficiency of si-CD147. Confocal microscopy was used to determine the effects of si-CD147 on SMMC-7721 cells' cytoskeleton. Invasion assay, gelatin zymography and cell adhesion assay were employed to investigate the effects of si-CD147 on SMMC-7721 cells' invasion, gelatinase production and cell adhesive abilities. Western blot assay was utilized to detect the effects of si-CD147 on focal adhesion kinase (FAK), vinculiln and mitogen-activated protein kinase (MAPK) expression in SMMC-7721 cells.
Downregulation of CD147 gene induced the alteration of SMMC-7721 cell cytoskeleton including actin, microtubule and vimentin filaments, and inhibited gelatinase production and expression, cells invasion, FAK and vinculin expression. si-CD147 also blocked SMMC-7721 cells adhesion to collagen IV and phosphorylation level of SAPK/JNKs. SAPK/JNKs inhibitor SP600125 inhibited gelatinase production and expression.
CD147 is required for normal tumor cell architecture and cell invasion. Downregulation of CD147 affects HCC cell structure and function. Moreover, the alteration of cell behavior may be related to SAPK/JNK Pathway. siRNA against CD147 may be a possible new approach for HCC gene therapy.
PMCID: PMC2572039  PMID: 18847500
25.  Growth inhibition and apoptosis induced by 6-fluoro-3-formylchromone in hepatocellular carcinoma 
BMC Gastroenterology  2014;14:62.
Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers in human population. The 6-fluoro-3-formylchromone (FCC) has been shown to have anti-tumor activity against various tumor cells. However, the effects of FCC on HCC cell lines have not yet been reported. This study aims to research the effects of FCC on HCC and advance the understanding of the molecular mechanism.
HCC cell line SMMC-7721 was treated with FCC at various concentrations (0, 2, 5, 10, and 20 μg/ml) for 24, 48 and 72 h, respectively. The proliferations of SMMC-7721 cells were measured by MTT assays. After cultured 24 hours, cell cycle distribution and apoptosis were determined by flow cytometry. However, the expression levels of PCNA, Bax and Bcl-2 were measured by western blotting after 48 hours.
FCC displayed a dose- and time-dependent inhibition of the SMMC-7721 cell proliferations in vitro. It also induced apoptosis with 45.4% and caused cell accumulation in G0/G1 phase with 21.5%. PCNA and Bcl-2 expression was significantly suppressed by FCC in a dose-dependent manner (P < 0.05), while Bax expression was increased.
FCC could significantly inhibit HCC cell growth in vitro through cell cycle arrest and inducing apoptosis by suppressing PCNA expression and modulating the Bax/Bcl-2 ratio.
PMCID: PMC4005831  PMID: 24708487
FCC; Cell Proliferation; Apoptosis

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