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
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.
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
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
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.
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
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.
Hepatocellular carcinoma; Ezrin-radixin-moesin-binding phosphoprotein-50; Growth; Migration; Invasion
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.
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.
The multi-kinase inhibitor Sorafenib, is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Apoptosis has been shown to be induced in HCC by several agents, including Sorafenib, as well as by the naturally occurring K vitamins (VKs). Since few non toxic agents have activity against HCC growth, we evaluated the activity of Sorafenib and K vitamins, both independently and together on the growth in vitro and in vivo of HCC cells. We found that when VK was combined with Sorafenib, the concentration of Sorafenib required for growth inhibition was substantially reduced. Conversely, VK enhanced Sorafenib effects in several HCC cell lines on growth inhibition. Growth could be inhibited at doses of VK plus Sorafenib that were ineffective with either agent alone,using vitamins K1, K2 and K5. Combination VK1 plus Sorafenib induced apoptosis on FACS, TUNEL staining and caspase activation. Phospho-ERK levels were decreased, as was Mcl-1, an ERK target. Sorafenib alone inhibited growth of transplantable HCC in vivo. At sub-effective Sorafenib doses in vivo, addition of VK1 caused major tumor regression, with decreased phospho-ERK and Mcl-1 staining. Thus, combination VK1 plus Sorafenib strongly induced growth inhibition and apoptosis in rodent and human HCC and inhibited the RAF/MEK/ERK pathway. VK1 alone activated PKA, a mediator of inhibitory Raf phosphorylation. Thus, each agent can antagonize Raf; Sorafenib as a direct inhibitor and VK1 through inhibitory Raf phosphorylation. Since both agents are available for human use, the combination has potential for improving Sorafenib effects in HCC.
Sorafenib; Vitamin K; Apoptosis; ERK; HCC
Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25− effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determineimmune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings showthat sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.
Sorafenib; T cell; Regulatory T cells; Hepatocellular carcinoma; HCV
Hepatocellular carcinoma (HCC) is one of the most commonly diagnosed tumors worldwide and is known to be resistant to conventional chemotherapy. New therapeutic strategies are urgently needed for treating HCC. Osthole, a natural coumarin derivative, has been shown to have anti-tumor activity. However, the effects of osthole on HCC have not yet been reported.
Methods and Findings
HCC cell lines were treated with osthole at various concentrations for 24, 48 and 72 hours. The proliferations of the HCC cells were measured by MTT assays. Cell cycle distribution and apoptosis were determined by flow cytometry. HCC tumor models were established in mice by subcutaneously injection of SMMC-7721 or Hepa1-6 cells and the effect of osthole on tumor growths in vivo and the drug toxicity were studied. NF-κB activity after osthole treatment was determined by electrophoretic mobility shift assays and the expression of caspase-3 was measured by western blotting. The expression levels of other apoptosis-related genes were also determined by real-time PCR (PCR array) assays. Osthole displayed a dose- and time-dependent inhibition of the HCC cell proliferations in vitro. It also induced apoptosis and caused cell accumulation in G2 phase. Osthole could significantly suppress HCC tumor growth in vivo with no toxicity at the dose we used. NF-κB activity was significantly suppressed by osthole at the dose- and time-dependent manner. The cleaved caspase-3 was also increased by osthole treatment. The expression levels of some apoptosis-related genes that belong to TNF ligand family, TNF receptor family, Bcl-2 family, caspase family, TRAF family, death domain family, CIDE domain and death effector domain family and CARD family were all increased with osthole treatment.
Osthole could significantly inhibit HCC growth in vitro and in vivo through cell cycle arrest and inducing apoptosis by suppressing NF-κB activity and promoting the expressions of apoptosis-related genes.
Sorafenib, a multi-tyrosine kinase inhibitor, is a standard treatment for advanced hepatocellular carcinoma (HCC). The present study was undertaken to determine whether the growth and metastasis of HCC were influenced in mice receiving sorafenib prior to implantation with tumors, and to investigate the in-vivo and in-vitro effect of sorafenib on natural killer (NK) cells. In sorafenib-pretreated BALB/c nu/nu mice and C57BL/6 mice, tumor growth was accelerated, mouse survival was decreased, and lung metastasis was increased. However, the depletion of NK1.1+ cells in C57BL/6 mice eliminated sorafenib-mediated pro-metastatic effects. Sorafenib significantly reduced the number of NK cells and inhibited reactivity of NK cells against tumor cells, in both tumor-bearing and tumor-free C57BL/6 mice. Sorafenib down-regulated the stimulatory receptor CD69 in NK cells of tumor-bearing mice, but not in tumor-free mice, and inhibited proliferation of NK92-MI cells, which is associated with the blocking of the PI3K/AKT pathway, and inhibited cytotoxicity of NK cells in response to tumor targets, which was due to impaired ERK phosphorylation. These results suggest immunotherapeutic approaches activating NK cells may enhance the therapeutic efficacy of sorafenib in HCC patients.
AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells.
METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line (LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, respectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA targeting ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analyses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) assay (in vitro) and tumour-growth assay (in vivo), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro).
RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest level of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expression was effectively inhibited (about 80%) by ANXA2-speciﬁc small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cellular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shRNA-mediated ANXA2 silencing in vitro, and the tumour growth inhibition rate was 38.24% in vivo. The percentage of MHCC97-H cells in S phase dramatically decreased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells decreased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis.
CONCLUSION: shRNA-mediated silencing of ANXA2 suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.
Annexin A2; Small hairpin RNA; Hepatocellular carcinoma; Invasion; Migration; Tumorigenic potential
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.
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
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.
Insufficient radiofrequency ablation; Epithelial-mesenchymal transition; Hepatocellular carcinoma; Metastasis
The growth inhibitory effects of D-glucosamine hydrochloride (GlcNH2·HCl), D-glucosamine (GlcNH2) and N-acetyl glucosamine (NAG) on human hepatoma SMMC-7721 cells in vitro were investigated. The results showed that GlcNH2·HCl and GlcNH2 resulted in a concentration-dependent reduction in hepatoma cell growth as measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. This effect was accompanied by a marked increase in the proportion of S cells as analyzed by flow cytometry. In addition, human hepatoma SMMC-7721 cells treated with GlcNH2·HCl resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis. NAG could not inhibit the proliferation of SMMC-7721 cells. GlcNH2·HCl exhibited antitumor activity against Sarcoma 180 in Kunming mice at dosage of 125~500 mg/kg, dose of 250 mg/kg being the best. GlcNH2·HCl at dose of 250 mg/kg could enhance significantly the thymus index, and spleen index and could promote T lymphocyte proliferation induced by ConA. The antitumor effect of GlcNH2·HCl is probably host-mediated and cytocidal.
D-glucosamine hydrochloride; D-glucosamine; N-acetyl glucosamine; Antitumor; Human hepatoma cell; Sarcoma 180
Two novel cyclodepsipeptides containing an unusual anthranilic acid dimer and a d-phenyllactic acid residues, clavatustides A (1) and B (2), were identified from cultured mycelia and broth of Aspergillus clavatus C2WU isolated from Xenograpsus testudinatus, which lives at extreme, toxic habitat around the sulphur-rich hydrothermal vents in Taiwan Kueishantao. This is the first example of cyclopeptides containing an anthranilic acid dimer in natural products, and the first report of microbial secondary metabolites from the hydrothermal vent crab. Clavatustides A (1) and B (2) suppressed the proliferation of hepatocellular carcinoma (HCC) cell lines (HepG2, SMMC-7721 and Bel-7402) in a dose-dependent manner, and induced an accumulation of HepG2 cells in G1 phase and reduction of cells in S phase.
cyclodepsipeptide; Aspergillus clavatus; hydrothermal vent; carcinoma cycle inhibitory
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.
Autophagy, a self-defense mechanism, has been found to be associated with drug resistance in hepatocellular carcinoma (HCC). Our study was designed to investigate the role and related mechanisms of autophagy in matrine-induced apoptosis in hepatoma cells of HepG2 and Bel7402. Cell apoptosis was detected by flow cytometry analysis (Annexin V–FITC/PI double-staining assay), the activity and activating cleavages of caspase-3, -8, and -9. MTT assay and colony forming assay were used to assess the effect of matrine on growth and proliferation of HCC cells. Autophagic flux in HCC cells was analyzed using the expression of LC3BI/II and p62/SQSTM1, GFP-LC3 transfection, and transmission electron microscopy. Moreover, regarding to the associated mechanisms, the effects of matrine on the phosphoinositide 3-kinase/AKT/mTOR pathway and beclin-1 were studied. Our results showed that: (1) both autophagy and apoptosis could be induced by treatment with matrine; (2) using the autophagic inhibitor chloroquine and beclin-1 small-interfering RNA, cell apoptosis induced by matrine could be enhanced in a caspase-dependent manner; and (3) autophagy was induced via inhibition of PI3K/AKT/mTOR pathway and up-regulation of beclin-1. In conclusion, inhibition of autophagy could enhance matrine-induced apoptosis in human hepatoma cells.
matrine; hepatocellular carcinoma; autophagy; apoptosis; autophagic inhibitor; chloroquine
Hepatocellular carcinoma (HCC) is one of the most common solid cancers, representing the third cause of cancer-related death among cirrhotic patients. Treatment of advanced HCC has become a very active area of research. Perifosine, a new synthetic alkylphospholipid Akt inhibitor, has shown anti-tumor activity by inhibition of Akt phosphorylation. In this study, the effect of perifosine on the cell proliferation and apoptosis in hepatoma cells has been investigated. Cell growth inhibition was detected by MTT assay, cell cycle was analyzed by flow cytometry, AnnexinV-FITC apoptosis detection kit was used to detect cell apoptosis, and protein expression was examined by Western blotting analysis. Our present studies showed that Akt phosphorylation was inhibited by perifosine in HepG2 and Bel-7402 human hepatocellular carcinoma cells. Perifosine inhibited the growth of HepG2 cells and Bel-7402 cells in a dose-dependent manner, and arrested cell cycle progression at the G2 phase. Apoptosis induction became more effective with increasing perifosine concentration. The caspase cascade and its downstream effectors, Poly (ADP-ribose) polymerase (PARP), were also activated simultaneously upon perifosine treatment. The proapoptotic effect of perifosine was in part depending on regulation of the phosphorylation level of ERK and JNK. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in HepG2 cells. Down-regulating the expression of Bcl-2 and up-regulating the level of Bax may be the potential mechanism for this synergistic effect. Our findings suggest that the small molecule Akt inhibitor perifosine shows substantial anti-tumor activity in human hepatoma cancer cell lines, and is a good candidate for treatment combinations with classical cytostatic compounds in hepatocellular carcinoma.
Hepatocellular carcinoma; Perifosine; PI3 K/Akt; Apoptosis; Caspase; Bcl-2
We investigated the molecular mechanisms underlying the effect of sorafenib and SC-59, a novel sorafenib derivative, on hepatocellular carcinoma (HCC). Sorafenib activated autophagy in a dose- and time-dependent manner in the HCC cell lines PLC5, Sk-Hep1, HepG2 and Hep3B. Sorafenib downregulated phospho-STAT3 (P-STAT3) and subsequently reduced the expression of myeloid cell leukemia-1 (Mcl-1). Inhibition of Mcl-1 by sorafenib resulted in disruption of the Beclin 1-Mcl-1 complex; however, sorafenib did not affect the amount of Beclin 1, suggesting that sorafenib treatment released Beclin 1 from binding with Mcl-1. Silencing of SHP-1 by small interference RNA (siRNA) reduced the effect of sorafenib on P-STAT3 and autophagy. Ectopic expression of Mcl-1 abolished the effect of sorafenib on autophagy. Knockdown of Beclin 1 by siRNA protected the cells from sorafenib-induced autophagy. Moreover, SC-59, a sorafenib derivative, had a more potent effect on cancer cell viability than sorafenib. SC-59 downregulated P-STAT3 and induced autophagy in all tested HCC cell lines. Furthermore, our in vivo
data showed that both sorafenib and SC-59 inhibited tumor growth, downregulated P-STAT3, enhanced the activity of SHP-1 and induced autophagy in PLC5 tumors, suggesting that sorafenib and SC-59 activate autophagy in HCC. In conclusion, sorafenib and SC-59 induce autophagy in HCC through a SHP-1-STAT3-Mcl-1-Beclin 1 pathway.
SC-59; sorafenib; STAT3; HCC
Sorafenib increases survival rate of patients with advanced hepatocellular carcinoma (HCC). The mechanism underlying this effect is not completely understood. In this work we have analyzed the effects of sorafenib on autocrine proliferation and survival of different human HCC cell lines. Our results indicate that Sorafenib in vitro counteracts autocrine growth of different tumor cells (Hep3B, HepG2, PLC-PRF-5, SK-Hep1). Arrest in S/G2/M cell cycle phases were observed coincident with cyclin D1 down-regulation. However, sorafenib’s main anti-tumor activity seems to occur through cell death induction which correlated with caspase activation, increase in the percentage of hypodiploid cells, activation of BAX and BAK and cytochrome c release from mitochondria to cytosol. In addition, we observed a rise in mRNA and protein levels of the pro-apoptotic “BH3-domain only” PUMA and BIM, as well as decreased protein levels of the anti-apoptotic MCL1 and survivin. PUMA targeting knockdown, by using specific siRNAs, inhibited sorafenib-induced apoptotic features. Moreover, we obtained evidence suggesting that sorafenib also sensitizes HCC cells to the apoptotic activity of Transforming Growth Factor-β (TGF-β) through the intrinsic pathway and to Tumor Necrosis Factor-α (TNF) through the extrinsic pathway. Interestingly, sensitization to sorafenib-induced apoptosis is characteristic of liver tumor cells, since untransformed hepatocytes did not respond to sorafenib inducing apoptosis, either alone or in combination with TGF-β or TNF. Indeed, sorafenib effectiveness in delaying HCC late progression might be partly related to a selectively sensitization of HCC cells to apoptosis by disrupting autocrine signals that protect them from adverse conditions and pro-apoptotic physiological cytokines.
TNF; TGF-beta; hepatocytes; PUMA; cancer
Sorafenib is currently approved for advanced hepatocellular carcinoma (HCC) and is presently being studied as an adjuvant treatment for HCC following resection. The effects of sorafenib on liver regeneration have not been clearly defined. Our objective was to identify the effects of sorafenib on liver regeneration in a murine partial hepatectomy (PH) model.
Materials and Methods
We performed PH in C57Bl/6 mice treated with a range of sorafenib doses with assessments at several time points. Liver sinusoidal endothelial cells (LSEC) and hepatocyte DNA synthesis and proliferation were assessed with 5-bromo-2-deoxyuridine (BrDU) and Ki67 by flow cytometry and immunohistochemistry (IHC).
Treatment with sorafenib did not result in any deaths following PH. When we measured BrDU uptake to assess DNA synthesis, there was a statistically significant increase at 48 HR post-PH for non-fibrotic LSEC following treatment with 60 mg/kg of sorafenib. However, BrDU and Ki67 staining among LSEC and hepatocytes was not significantly affected by sorafenib at any of the other doses or time points. BrDU and Ki67 flow cytometry data correlated with IHC findings and post-operative liver weights.
In a murine PH model, sorafenib did not alter the repair response of normal or fibrotic livers following PH as measured by changes in liver weight, DNA synthesis, and cellular proliferation. These findings suggest sorafenib administered following hepatic resection does not impair liver regeneration.
Sorafenib; liver regeneration; partial hepatectomy; BrdU; Ki67