The aim of the present study was to evaluate whether downregulation of extracellular signal-regulated kinase 1/2 (ERK1/2) is involved in conventional reversal methods and whether the inhibitors of the ERK signaling pathway reverse multidrug resistance (MDR) in hepatocellular carcinoma (HCC) cells. The sensitivities of SMMC7721 and BEL7402, and the MDR SMMC7721/Adriamycin (ADM) and BEL7402/ADM HCC cell lines to ADM were evaluated by CellTiter-Glo® luminescent cell viability assay through calculating the half maximal inhibitory concentration (IC50) of ADM. In addition, the expression levels of ERK1/2 and phosphorylated (p)ERK1/2 were determined by western blot analysis subsequent to treatment of the cells with PD98059, an MEK inhibitor, or sorafenib, a multikinase inhibitor. The results revealed that the ADM IC50 for the SMMC7721/ADM cells was 16.44 times higher than that of the SMMC7721 cells (P<0.05), and the ADM IC50 for the BEL7402/ADM cells was 20.34 times higher than that of the BEL7402 cells (P<0.05). Following treatment with PD98059 or sorafenib, the expression levels of pERK1/2 in the MDR cells decreased in a dose-dependent manner. Subsequent to treatment with 5 μM PD98059, the ADM IC50 values for the SMMC7721/ADM and BEL7402/ADM cells were reduced to 0.8±0.056 and 1.583±0.284 μg/ml, respectively. Following treatment with 2.5 μM sorafenib, the ADM IC50 values for the SMMC7721/ADM and BEL7402/ADM cells were reduced to 0.264±0.049 and 1.099±0.135 μg/ml, respectively. Subsequent to incubation with 4 μg/ml cyclosporine A (CsA), a classic MDR reversal agent, the ADM IC50 values in the SMMC7721/ADM and BEL7402/ADM cells were reduced to 0.349±0.023 and 0.427±0.039 μg/ml, respectively. CsA treatment also increased the expression levels of pERK1/2 without affecting the total ERK1/2 levels. Therefore, the inhibition of ERK signaling pathway activity may be an important method to reverse the MDR of HCC cells, but is not unique.
hepatocellular carcinoma; multidrug resistance; reverse; extracellular signal-regulated kinase/mitogen-activated protein kinase signaling pathway
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.
Hepatocellular carcinoma; Sorafenib; 5-fluorouracil; Cell cycle arrest
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.
Abstract: Purpose: To investigate whether fructopyrano-(1→4)-glucopyranose (FG) inhibits the proliferation of liver cancer cells and angiogenesis in a vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) dependent manner. Methods: Bel-7402, HepG2 and SMMC-7721 cells with high expression of VEGF and VEGFR were screened. Bel-7402 cells and human microvascular endothelial cells (HMEC) were treated with FG for 48 h. CCK-8 assay was used to detect cell proliferation. Wound healing assay was used to investigate effect of FG on the migration of HMECs. Tube formation assay was done to test influence of FG on the angiogenesis of HMECs, and qRT-PCR and western blot assay were performed to detect mRNA and protein expression of VEGF, Fit-1 and KDR. Nude mice were inoculated with Bel-7402 cells, and influence of FG on tumor growth, microvessel density (MVD) and VEGF expression in tumor was investigated. Results: Bel-7402 cells had a significantly higher expression of VEGF and VEGFR when compared with HepG2 cells and SMMC-7721 cells. FG could markedly reduce the mRNA and protein expressions of VEGF, Fit-1 and KDR in Bel-7402 cells and inhibit the proliferation of Bel-7402 cells in a concentration dependent manner. In addition, FG was able to remarkably inhibit the proliferation, migration and angiogenesis of HMECs, exerting anti-angiogenetic effect. In cancer-bearing nude mice, FG was found to inhibit the tumor growth, reduce MVD in tumors and decrease the VEGF in tumors. Conclusions: FG can inhibit proliferation of liver cancer cells and suppression angiogenesis in liver cancer in a VEGF/VEGFR dependent manner.
Vascular endothelial growth factor; vascular endothelial growth factor receptor; liver cancer; fructopyrano-(1→4)-glucopyranose
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.
YC-1; Sorafenib; Hepatocellular carcinoma; STAT3
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.
Death receptor 3 (DR3) belongs to the tumor necrosis factor (TNF) receptor superfamily, primarily found in lymphoid tissues. Reports have determined that DR3 may also be distributed in numerous types of tumors. Therefore, it is thought that DR3 may have an important role in the process of tumorigenesis. The aim of the present study was to observe the effect of silencing DR3 expression on hepatocarcinoma cell growth, apoptosis and invasion in order to elucidate the role of DR3 in tumor development. The hepatocarcinoma cell lines (HepG2, Huh7, SMMC7721 and Bel-7402) and normal human liver cells (HL-7702) were transfected with three stealth RNA interference (RNAi) sequences that target the DR3 gene. Reverse transcription quantitative polymerase chain reaction was used to detect the expression levels of DR3 in hepatocarcinoma cell lines and normal liver HL-7702 cells. MTT assay and flow cytometry (FCM) were used to determine the rates of cell proliferation and apoptosis, respectively. Following silencing of the DR3 gene, western blot analysis was used to determine the protein expression of P53, Fas, Caspase8, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Caspase3. DR3 messenger RNA (mRNA) expression in hepatocarcinoma cell lines was significantly increased compared with that in the normal liver cell line. Three targeted DR3 gene small interfering RNAs significantly inhibited DR3 gene expression in Bel-7402 cells at the nucleic acid level. AF02670.1_stealth_883 and cocktail demonstrated the most efficient inhibition of DR3 gene expression at 48 and 72 h following transfection, with mRNA inhibition rates of 89.46 and 92.75%, and 90.53 and 94.25% (P<0.01), respectively. Cell viability was significantly reduced by AF02670.1_stealth_883 and RNAi cocktail at 24, 48 and 72 h following transfection. The inhibition rates of cell proliferation were 50.76 and 61.76% (P<0.05) at 72 h following transfection. FCM revealed that AF02670.1_stealth_883 and RNAi cocktail also induced apoptosis in Bel-7402 cells at 72 h following transfection. Reduction of NF-κB and P53 levels was observed (P<0.05) in Bel-7402 cells following DR3 silencing, whereas levels of Fas, Caspase3 and Caspase8 were markedly elevated (P<0.05). DR3 expression levels in hepatocellular carcinoma cells were significantly higher than those in normal cells. DR3 silencing effectively inhibited proliferation and invasion of hepatocellular carcinoma cells in vitro. However, silencing of the DR3 gene affect levels of apoptosis antigen-3 ligand in cells, therefore indicating that it may be involved with other pathways that regulate apoptosis in HCCs. In conclusion, the results of the present study indicated that DR3 may be a promising therapeutic target molecule for further study of hepatocellular carcinoma gene therapy.
hepatocellular carcinoma; death receptor 3; RNA interference; apoptosis; caspase3; apoptosis antigen-3 ligand
AIM: To investigate the cyclooxygenase-2 (COX-2) expression level in human HepG2, Bel-7402 and SMMC-7721 hepatoma cell lines and the molecular mechanism of COX-2 selective inhibitor celecoxib-induced cell growth inhibition and cell apoptosis.
METHODS: Hepatoma cells were cultured and treated with celecoxib. Cell in situ hybridization (ISH) and immunocytochemistry were used to detect COX-2 mRNA and protein expression. Proliferating cell nuclear antigen and phosphorylated Akt were also detected by immunocytochemistry assay. Cell growth rates were assessed by 3-(4, 5-dimethylthiazol-2-yl-2, 5-diphenylte-trazolium (MTT) bromide colorimetric assay. Celecoxib-induced cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and flow cytometry (FCM). The phosphorylated Akt and activated fragments of caspase-9, caspase-3 were examined by Western blotting analysis.
RESULTS: Increased COX-2 mRNA and protein expression were detected in all three hepatoma cell lines. Celecoxib could significantly inhibit cell growth and the inhibitory effect was in a dose- and time-dependent manner evidenced by MTT assays and morphological changes. The apoptotic index measured by TUNEL increased correspondingly with the increased concentration of celecoxib and the reaction time. With 50 μmol/L celecoxib treatment for 24 h, the apoptotic index of HepG2, BEL-7402 and SMMC-7721 cells was 25.01±3.08%, 26.40±3.05%, and 30.60±2.89%, respectively. Western blotting analysis showed remarkable activation of caspase-9, caspase-3 and dephosphorylation of Akt (Thr308). Immunocytochemistry also showed the reduction of PCNA expression and phosphorylation Akt (Thr308) after treatment with celecoxib.
CONCLUSION: COX-2 mRNA and protein overexpression in HepG2, Bel-7402 and SMMC-7721 cell lines correlate with the increased cell growth rate. Celecoxib can inhibit proliferation and induce apoptosis of hepatoma cell strains in a dose- and time-dependent manner.
Apoptosis; Akt; Celecoxib; Caspase; Cell proliferation; COX-2; HCC; PCNA
Targeting protein for Xenopus kinesin-like protein 2 (TPX2) is a nuclear proliferation-related protein that plays a critical role in the formation of mitotic spindle. High expression of TPX2 has been observed in several types of tumors. However, the role of TPX2 in hepatocellular carcinoma (HCC) remains unclear. Our study aimed to investigate the effect of TPX2 on HCC cell invasion.
The immortalized normal human liver cell line L02 and six HCC cell lines including SMMC-7721, BEL-7402, Huh-7, HepG2, Hep3B and SKHep1 were subjected to qRT-PCR and western blot for TPX2 mRNA and protein, respectively. Furthermore, TPX2 small interfering RNA (siRNA) was used to knock down TPX2 expression in SMMC-7721 and HepG2 cells. Cell proliferation and invasion were determined by MTT and transwell assays. Otherwise, expression of p-AKT, MMP2 and MMP9 were evaluated by western blot in SMMC-7721 cells.
The expression of TPX2 in HCC cell lines was markedly higher than that in normal human liver cell line. TPX2 knockdown using a specific TPX2-siRNA reduced the number of invaded cells and inhibited cell proliferation in SMMC-7721 and HepG2 cells. Furthermore, TPX2 knockdown resulted in inactivation of AKT signaling and down-regulation of MMP2 and MMP9 expression in SMMC-7721 cells.
Our study identified that TPX2 might contribute to tumor cell invasion through activating AKT signaling and subsequently increasing MMP2 and MMP9 in HCC.
Targeting protein for Xenopus kinesin-like protein 2 (TPX2); hepatocellular carcinoma; MMP2; MMP9
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.
sorafenib; all-trans retinoic acid; growth inhibition; gap junction; hepatocellular carcinoma
AIM: To determine the radiosensitizing potential of docetaxel in human hepatocellular carcinoma SMMC-7721 cells and its mechanisms.
METHODS: SMMC-7721 cells were incubated with docetaxel at 0.125, 0.25, and 0.5 nmoL/L for 24 h and at 0.125 and 0.25 nmol/L for 48 h before irradiation. Radiation doses were given from 0 to 10 Gy. Cell survival was measured by a standard clonogenic assay after a 9-d incubation. The reactive oxygen species (ROS) and glutathione (GSH) are detected after being given the same dose of docetaxel for the same time.
RESULTS: The sensitization enhancement ratios (SER) for SMMC-7721 cells determined at the 50% survival level were 1.15, 1.21 and 1.49 at 0.125, 0.25, and 0.5 nmol/L for pre-incubation of 24 h, respectively; the SER were 1.42, 1.67 at 0.125 and 0.25 nmol/L, for pre-incubation of 48 h, respectively. The ROS of SMMC-7721 cells increased and GSH decreased after pretreatment with the same doses of docetaxel for 24 or 48 h.
CONCLUSION: A radiosensitizing effect of docetaxel could be demonstrated unambiguously in this cell line used. In addition, our data showed that the mechanism of radiopotentiation by docetaxel probably does not involve a G2/M block in SMMC-7721 cells, and ROS generation and GSH deletion may play a key role in the radiosensitizing effect of docetaxel.
Docetaxel; Hepatocellular carcinoma; SMMC-7721cell line; Radiosensitization; Reactive oxygen species
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. (ClinicalTrials.gov number, NCT00999843.)
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.
Hepatocellular carcinoma; sall3; Aberrant methylation; Down regulation mRNA transcription
Oncolytic herpes simplex virus (HSV) can replicate in and kill cancer cells while sparing the adjacent normal tissue. Hepatocellular carcinoma (HCC) is amongst the most common and lethal cancers, especially in Third World countries. In this study, the cytotoxicity of a third-generation oncolytic HSV, G47Δ, was investigated in different human HCC cell lines and in an immortalized human hepatic cell line. Additionally, subcutaneous models of HCC were established to evaluate the in vivo anti-tumor efficacy of G47Δ.
The HepG2, HepB, SMMC-7721, BEL-7404, and BEL-7405 human HCC cell lines and the HL-7702 human hepatic immortalized cell lines were infected with G47Δ at different multiplicities of infection (MOIs). The viability of infected cells was determined, and the G47Δ replication was identified by X-gal staining for LacZ expression. Two subcutaneous (s.c.) HCC tumor models of HCC were also established in Balb/c nude mice, which were intratumorally(i.t.) treated with either G47Δ or mock virus. Tumor volume and mouse survival times were documented.
More than 95% of the HepG2, Hep3B,and SMMC-7721 HCC cells were killed on by day 5 after infection with a MOI’s of 0.01. For the HL-7702 human hepatic immortalized cells, 100% of the cells were killed on by day 5 after infection with a MOI’s of 0.01. The BEL-7404 HCC cell line was less susceptible with about 70% cells were killed by day 5 after infection with a MOI’s of 0.01. Whereas the BEL-7405 HCC cells were the least susceptible, with only 30% of the cells were killed. Both the SMMC-7721 and BEL-7404 cells form aggressive sc tumor models. G47Δ replicates in the tumors, such that most of the tumors regressed after the G47Δ-treatment, and treated tumor-bearing mice survived much longer than the control animals.
G47Δ effectively kills human HCC cells and an immortalized hepatic cell line at low MOI. Intra-tumor injection of G47Δ can induce a therapeutic effect and prolong the survival of treated mice bearing SMMC-7721 and BEL-7404 subcutaneously (s.c.) tumors. Thus, G47Δ may be useful as a novel therapeutic agent for HCC.
Hepatocellular carcinoma; Oncolytic herpes simplex virus; Cytotoxicity; Subcutaneous model
To investigate the anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in hepatocellular carcinoma (HCC) cell lines.
HCC cell lines BEL7402, SMMC-7721, MHCC97L, MHCC97H, and MHCCLM3 were used. HCC cells were treated with dsP21-322 (50 nmol/L), dsControl (50 nmol/L), siP21 (50 nmol/L), or mock transfection. The expression of p21 was detected using quantitative PCR and Western blot. The effects of RNA activation on HCC cells were determined using cell viability assays, apoptosis analyses and clonogenic survival assays. Western blot was also conducted to detect the expression of Bcl-xL, survivin, cleaved caspase-3, cleaved caspase-9 and cleaved PARP.
At 72 to 120 h following the transfection, dsP21-322 markedly inhibited the viability of HCC cells and clone formation. At the same times, dsP21-322 caused a significant increase in HCC cell apoptosis, as demonstrated with cytometric analysis. The phenomena were correlated with decreased expression levels of the anti-apoptotic proteins Bcl-xL, surviving, and increased expression of cleaved caspase-3, cleaved caspase-9 and cleaved PARP.
RNA-induced activation of p21 gene expression may have significant therapeutic potential for the treatment of hepatocellular carcinoma and other cancers.
hepatocellular carcinoma; small activating RNA (saRNA); RNA-induced gene activation (RNAa); p21WAF1/CIP1; cell viability; apoptosis
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.
Hepatocellular carcinoma; 8-bromo-7-methoxychysin; Chrysin; Reactive oxygen species; Jun N-terminal kinase
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.
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.
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.
Multidrug resistance; Extracellular signal-regulated MAP kinases; Hepatocellular carcinoma; P-glycoprotein; Multidrug resistance-associated protein
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.
radiation; sorafenib; colorectal cancer; DNA damage; cell cycle
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.
Zuojinwan (ZJW), a famous Chinese medicinal formula, contains two medicinal herbs Coptis chinese Frach and Evodia rutaecarpa (Juss.) Benth in the ratio of 6: 1. The inhibitory effects of ZJW on eight kinds of human cancer cell lines including SMMC-7721, BEL-7402, BEL-7404, HepG2, A549, NCI-H446, NCI-H460 and HCT- 116 cells were evaluated, and the possible mechanism was investigated. The growths of the eight kinds of cancer cells were inhibited by ZJW assessed through MTT assay. Flow cytometry assay revealed a sub-G1 peak with reduced DNA content was formed. The cell cycle was arrested in the G0/G1 phase in ZJW-treated SMMC-7721 and HepG2 cells, and in the S phase for NCI-H460 cells. Significant DNA damage was produced by ZJW assessed with single-cell gel electrophoresis assay. Morphological changes were also observed. Caspase-3 and -9 activities were increased following ZJW treatment. Western blot analysis showed that Bax and Bak protein levels were increased after ZJW treatment, while Bcl-2 and Bcl-xl protein levels were decreased. Our results suggest that ZJW has significant anti-cancer activities due to induction of mitochondria- dependent apoptosis pathway. Therefore, ZJW has the potential to be a novel chemotherapy drug to treat hepatoma, lung cancer and colon cancer by suppressing tumor growth.
Apoptosis; Caspase; Cell cycle; Mitochondrial pathway; Zuojinwan
AIM: To investigate the cytotoxicity of the cytokine-induced killer (CIK) cells from the post-operation patients with primary hepatocellular carcinoma (HCC) to multidrug-resistant (MDR) cell of HCC both in vitro and in vivo.
METHODS: A drug-resistant cell line was established by culturing human HCC cell line Bel-7402 in complete RPMI 1640 medium with increasing concentrations of adriamycin from 10 to 2000 nmol/L. CIK cells were obtained by inducing the peripheral blood mononuclear cells with rhIFN-γ, monoclonal anti-CD3 antibody, rhIL-1α as well as rhIL-2, which were added into the culture. To detect the cytotoxicity of the CIK cells from HCC patients, the Bel-7402/R was taken as target (T) cells and CIK cells as effect (E) cells. Cytotoxic test was performed and measured by MTT. As to in vivo test, CIK cells were transfused into patients with HCC. The tumor specimens of the patients were obtained and immunohistochemistry was carried out to detect CD3, CD45, CD45RO as well as CD68.
RESULTS: A MDR 1 HCC cell line Bel-7402/R was established. Its MDR1 mRNA overexpressed which was shown by RT-PCR; the P-glycoprotein expression increased from 1.32% of parent cells to 54%. CIK cells expanded vigorously by more than 70-fold and the CD3+CD56+ increased by more than 600-fold after 3-wk incubation on average. The cytotoxicity of CIK from HCC patients to Bel-7402/R was about 50% and to L-02 below 10% (t = 8.87, P<0.01), the same as that of CIK from normal individuals. Each of the 17 patients received 1-5×1010 of CIK cell transfusion. No side effects were observed. After CIK treatment, the tumor tissue nodules formed and a large amount of lymphocytes infiltrated in the liver cancer tissue and CD3, CD45, CD45RO, and CD68 increased greatly which was shown by immunohistochemistry.
CONCLUSION: A stable MDR1 HCC cell line has been established which could recover from liquid nitrogen and CIK from HCC patients has strong cytotoxicity to MDR HCC cell. CIK adoptive immunotherapy is safe and has no side effects. Receivers improved their immunity to tumor evidently. CIK treatment may be a better choice for HCC patients after operation to prevent the recurrence, especially when tumors have developed drug resistance.
Hepatocellular carcinoma; Cytokine-induced killer; Cytotoxicity; Multidrug resistance; P-glycoprotein
The aim of this study was to investigate the effects of Saikosaponin-d (SSd) combined with radiotherapy on SMMC-7721 hepatoma cell lines and its mechanism.
SMMC-7721 hepatoma cell lines are selected in our research. With MTT (methylthiazolyldiphenyl-tetrazolium-bromide) method, the effects of SSd and radiation on inhibiting SMMC-7721 cell growth were investigated. We also used transmission electron microscopy (TEM) to observe ultrastructural changes of cells. Colorimetry methods were used to measure content changes of glutathione (GSH) and malondialdehyde (MDA) in cells.
Both SSd and radiation inhibited the growth of SMMC-7721 cells. The combination of SSd and radiotherapy had a time-dependent synergistic effect. Radiation caused ultrastructural damage to cells, and the damage was enhanced in combination with SSd. Radiation decreased the GSH content and increased the MDA content in cells, and this effect was suppressed after the intervention of SSd.
SSd can inhibit the growth of SMMC-7721 hepatoma cell lines in vitro. Additionally, it significantly enhances the effects of radiation on inhibiting the growth of SMMC-7721 hepatoma cell lines, and up-regulates the antioxidant level after the radiotherapy. Thus, SSd could be an ideal radiotherapy sensitizer for the treatment of liver cancer.
Saikosaponin-d; Radiation; Hepatocellular Carcinoma; Glutathione
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.